Objective·To study the effect of phosphatase and tensin homolog on chromosome 10 (PTEN) on alternative splicing of forkhead box M1 (FoxM1), and its impact on tumor cell migration.Methods·PTEN was knocked down by short hairpin RNA (shRNA) in human embryonic kidney 293T cells, human prostate cancer DU145 cells, human colorectal adenocarcinoma RKO cells, and human colon cancer SW480 and SW620 cells. Specific primers were designed for FoxM1 and its subtypes FoxM1B and FoxM1C, and the mRNA expression levels of FoxM1B and FoxM1C were detected by quantitative real-time PCR (qRT-PCR). FoxM1B and FoxM1C were overexpressed in DU145 cells, and their effects on tumor cell migration were tested by Transwell assay and wound healing assay. Immunofluorescence and dual luciferase reporter gene assay were used to explore the potential mechanism of differential regulation of tumor cell migration by FoxM1B and FoxM1C.Results·① PTEN was knocked down in 293T, DU145, RKO, SW480, and SW620 cell lines. qRT-PCR results showed that compared with the control cells, the mRNA expression level of FoxM1B significantly increased in PTEN knockdown cells, while the mRNA expression level of FoxM1C decreased or remained unchanged. Knockdown of PTEN did not affect the transcription level of FoxM1, but caused the variable splicing of FoxM1 and promoted the generation of FoxM1B. ② Compared with the control cells, the number of DU145 cells migrating to the below chamber increased in the FoxM1B overexpression group (P=0.024), while the number of migrating DU145 cells in the FoxM1C overexpression group was lower (P=0.000). The healing ability of DU145 cells was significantly enhanced in the FoxM1B overexpression group (P=0.001), while the healing ability of DU145 cells was weakened in the FoxM1C overexpression group (P=0.021). Overall, FoxM1B and FoxM1C had opposite effects on tumor cell migration. FoxM1B promoted tumor cell migration, while FoxM1C inhibited tumor cell migration. ③ Neither FoxM1B nor FoxM1C overexpression could induce β-catenin to enter the nucleus. Dual luciferase reporter gene assay showed no difference in the transcriptional activity of FoxM1B and FoxM1C. The difference between FoxM1B and FoxM1C in the regulation of tumor metastasis was also not mediated by β-catenin translocation.Conclusion·Knockdown of PTEN regulates the alternative splicing of FoxM1, leading to increasing expression of transcript FoxM1B, which plays a positive role in tumor cell migration.