Cancer is a deadly and fast-spreading disease that is a growing health problem worldwide due to a lack of comprehensive screening and appropriate medication. However, natural products derived from medicinal plants have gained attention as potential sources of bioactive compounds that selectively remove cancerous lesions and are nontoxic and safe. Pancreatic cancer (PC) is a major therapeutic challenge and is predicted to surpass breast cancer as the third leading cause of cancer death. This study investigated the cytotoxic effects of methanolic extracts from five Iranian medicinal plants, Cuscuta epithymum, Achillea millefolium, Salvia officinalis, Salvia hydrangea, and Teucrium polium, on pancreatic cancer cell lines (MIA PaCa-2 and PaTu8902). Additionally, we examined the changes in the expression of key genes following treatment with C. epithymum extract. The findings revealed that the plant extracts had a dose-dependent effect on the cell viability of the lines, with the C. epithymum extract exhibiting the greatest cytotoxic effect (IC50 values of 85.03 µg/mL for MIA PaCa-2 and 156.57 µg/mL for PaTu 8902). GC‒MS analysis revealed 25 bioactive compounds in C. epithymum, with quinic acid (14.13%), p-vinylphenol (13.22%), and valeraldehyde (11.21%) as the most abundant. The study also investigated the changes in the expression of the STAT4, PIK3CD, EMP1, and RAB11FIP3 genes in MIA PaCa-2 and PaTu 8902 pancreatic cancer cell lines after treatment with the extract from C. epithymum. A correlation was detected between the expression levels of PIK3CD, STAT4, EMP1, and RAB11FIP3 and various concentrations of C. epithymum extract. The results revealed that the extract increased the mRNA levels of STAT4, PIK3CD, and EMP1, whereas RAB11FIP3 was reduced in the treated cells. Accordingly, C. epithymum extract has strong cytotoxic effects on pancreatic cancer cells and influences the expression of key cancer-related genes, suggesting its potential as a therapeutic candidate for PC treatment. Further in vivo studies are recommended to explore its mechanisms of action and clinical applicability.