Background and purpose: Abnormal DNA methylation is closely associated with the onset and progression of tumors. This study aimed to investigate the expression of intermediate filament family orphan 1 (IFFO1), a methylation-driven gene (MDG) in pancreatic adenocarcinoma (PAAD), along with its effects on the invasion and metastasis of PAAD cells, as well as its potential as a diagnostic and prognostic biomarker. Methods: mRNA expression data (TCGA-PAAD-mRNA), DNA methylation data (TCGA-PAAD-meth, GSE53051, PACA-AU) of PAAD and adjacent normal tissues, as well as DNA methylation data of healthy individuals’ blood (GSE69270), were obtained from the The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO) databases. By performing differential expression analysis combined with differential methylation analysis, we screened for MDG in PAAD. In the TCGA database, Pearson correlation tests were employed to verify the relationship between IFFO1 promoter methylation level and its expression level. Additionally, Kaplan-Meier survival analysis was conducted to evaluate the relationship among IFFO1 promoter methylation level, expression level, and the prognosis of PAAD. Pathological sections of cancer tissues and corresponding adjacent tissues from 27 PAAD patients were obtained from Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine. All samples involved in this study were approved by the human ethics committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine (ethics number: hospital ethics review[2017]No.53). Immunohistochemistry staining (IHC) was utilized to detect the expression of IFFO1 in cancer tissues and corresponding adjacent tissues from 27 PAAD patients. Based on the median expression level of IFFO1, patients in the TCGA database were classified into high-expression and low-expression groups. Subsequently, differential analysis, gene ontology (GO) enrichment analysis and gene set enrichment analysis (GSEA) were performed. Western blot and real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) were employed to assess the expression variations of IFFO1 between the normal pancreatic ductal epithelial cell line H6C7 and the PAAD cell lines MIA PaCa2, BxPC-3, AsPC-1 and Capan-2. The impact of IFFO1 overexpression on the migration and invasion capacities of PAAD cell lines AsPC-1 and Capan-2 was evaluated using scratch and invasion assays. Additionally, receiver operating characteristic (ROC) curves and Kaplan-Meier survival analysis were utilized to assess the diagnostic and prognostic significance of IFFO1 methylation levels in the TCGA pan-cancer cohort. Results: Through the cross-screening of five datasets, 41 MDG in PAAD were identified. Among these, IFFO1 was found to be the gene most closely associated with the prognosis of PAAD [hazard ratio (HR)=0.28, P<0.001]. IFFO1 exhibited high methylation and low expression levels in PAAD. Moreover, a significant negative correlation was observed between the methylation level of its promoter and its expression level (r=-0.55, P<0.001). IHC results indicated that IFFO1 expression was significantly lower in PAAD tissues than in adjacent non-tumor tissues (P<0.05). TCGA survival analysis demonstrated that patients with high methylation or low expression of IFFO1 had poorer overall survival (P<0.05). Both GO and GSEA analyses indicated that the pathway “Negative regulation of cell migration” was enriched in patients with high IFFO1 expression. Western blot and RTFQ-PCR results demonstrated that IFFO1 expression in normal pancreatic ductal epithelial cells H6C7 was significantly higher compared with PAAD cell lines MIA PaCa2, BxPC-3, AsPC-1, and Capan-2. Overexpression of IFFO1 significantly inhibited the migration and invasion of the PAAD cell lines AsPC-1 and Capan-2. Additionally, pan-cancer analysis revealed that IFFO1 exhibited abnormal promoter methylation and low expression across various cancer types, with its methylation levels demonstrating significant diagnostic and prognostic prediction value among different tumors. Conclusion: Promoter hypermethylation results in decreased expression of IFFO1 in PAAD. IFFO1 may suppress the invasion and migration abilities of PAAD cells. Furthermore, IFFO1 methylation holds great promise as a novel biomarker for the diagnosis and prognosis of PAAD.