The transcription factor Forkhead box M1 (FOXM1) plays important roles in oncogenesis. However, the expression statuses of FOXM1 isoforms and their impact on and molecular basis in oncogenesis are unknown. We sought to determine the identities of FOXM1 isoforms in and the impact of their expression on pancreatic cancer development and progression using human tissues, cell lines, and animal models. Overexpression of FOXM1 mRNA and protein was pronounced in human pancreatic tumors and cancer cell lines. We identified five FOXM1 isoforms present in pancreatic cancer: FOXM1a, FOXM1b, and FOXM1c along with two isoforms tentatively designated as FOXM1b1 and FOXM1b2 because they were closely related to FOXM1b. Interestingly, FOXM1c was predominantly expressed in pancreatic tumors and cancer cell lines, whereas FOXM1a expression was generally undetectable in them. Functional analysis revealed that FOXM1b, FOXM1b1, FOXM1b2, and FOXM1c, but not FOXM1a, promoted pancreatic tumor growth and metastasis. Consistently, FOXM1b, FOXM1b1, FOXM1b2, and FOXM1c activated transcription of their typical downstream genes. Also, Sp1 mechanistically activated the FOXM1 promoter, whereas Krüppel-like factor 4 (KLF4) repressed its activity. Finally, we identified an Sp1- and KLF4-binding site in the FOXM1 promoter and showed that both Sp1 and KLF4 protein bound directly to it. Deletion mutation of this binding site significantly attenuated the transcriptional regulation of the FOXM1 promoter positively by Sp1 and negatively by KLF4. We showed that overexpression of specific FOXM1 isoforms critically regulates pancreatic cancer development and progression by enhancing tumor cell invasion and metastasis. Our findings strongly suggest that targeting specific FOXM1 isoforms effectively attenuates pancreatic cancer development and progression. Cancer Res; 73(13); 3987–96. ©2013 AACR.