This paper investigated whether 125I seed irradiation for pancreatic cancer treatment was mediated through the RBM15/KLF5 pathway in glycolysis. The study collected peripheral blood from pancreatic cancer patients, and detected the expression of RBM15 and KLF5 expression in the serum of pancreatic cancer patients before and after 125I seed irradiation. An in vitro study was conducted to investigate the effects 125I seed irradiation on the malignant behavior and glycolysis of pancreatic cancer cells. The underlying mechanisms were thoroughly examined through a series of logical experiments, including Western blot analysis, Dot-blot experiment, methylated RNA immunoprecipitation assay, and RNA pull down assay. A xenograft tumor model in nude mice was established and treated with 125I seed irradiation, which was employed to research the in vivo effect and mechanism of 125I seed irradiation for pancreatic cancer. The overexpressed RBM15 and KLF5 in serum of pancreatic cancer patients were reduced after 125I seed treatment. 125I seed treatment impaired pancreatic cancer cell proliferation and invasion; enhanced apoptosis; attenuated glycolysis; and reduced RBM15 and KLF5 expression. RBM15 overexpression partially reversed these influences of 125I seed treatment on pancreatic cancer cells. RBM15 was capable of increasing KLF5 expression, which might be achieved by promoting m6A methylation of KLF5. In vivo, 125I seed treatment blocked the growth of pancreatic cancer cells and decreased RBM15 and KLF5 expression in xenograft tumor, whereas RBM15 overexpression abolished these effects. 125I seed irradiation suppressed glycolysis in pancreatic cancer by inhibiting KLF5 m6A methylation through down-regulation of RBM15. This discovery established a solid foundation for the use of in the treatment of pancreatic cancer.