Background and Aims Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is essential in various tumors. However, the role of MTHFD1 in pancreatic cancer remains unclear. This study was conducted to explore the expression and clinical significance of MTHFD1 in pancreatic cancer through bioinformatics analysis and clinical sample validation, as well as to analyze its potential mechanisms of action in pancreatic cancer.Methods The GEPIA2 online platform was used to analyze the differential expression of MTHFD1, survival, and pathological stage in TCGA pancreatic cancer data, examining the relationship between MTHFD1 expression and clinicopathologic features of pancreatic cancer patients. Univariate and multivariate analyses were performed using the Cox proportional hazards model on TCGA data. GO, KEGG, and GSEA analyses were conducted to predict the possible mechanisms of MTHFD1 in pancreatic cancer. The expression of MTHFD1 in 80 cases of pancreatic cancer and adjacent tissues was detected using immunohistochemistry, qRT-PCR, and Western blot and its expression with clinicopathologic characteristics was analyzed.Results In the TCGA database, MTHFD1 expression in pancreatic cancer tissues was significantly higher than in normal tissues (P<0.05). High expression of MTHFD1 was significantly associated with poor prognosis in pancreatic cancer patients (P=0.007). TCGA data indicated a close correlation between MTHFD1 expression and tumor stage (P<0.05). MTHFD1 expression was identified as an independent prognostic factor for pancreatic cancer (HR=1.777, P=0.01). GO, KEGG, and GSEA analyses showed that MTHFD1 was related to the cell cycle, and correlation heatmaps indicated a strong association between the MTHFD1 gene and the cell cycle. In the TIMER database, MTHFD1 expression level was significantly correlated with various immune cells, including B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells (all P<0.05). The GDSC database revealed that patients with low MTHFD1 expression were more sensitive to various therapeutic agents than those with high expression. In clinical pancreatic cancer specimens, the positive expression rate of MTHFD1 and its mRNA and protein levels were significantly higher in cancer tissues than in adjacent tissues (all P<0.05). MTHFD1 expression was associated with tumor differentiation, clinical stage, lymph node metastasis, and neural infiltration (all P<0.05). Patients with high MTHFD1 expression had significantly shorter overall survival than those with low expression (P<0.05).Conclusion MTHFD1 is highly expressed in pancreatic cancer tissues and is associated with poor prognosis. It may participate in the occurrence and development of pancreatic cancer through the cell cycle and is related to the infiltration of tumor immune cells.