Increasing evidence suggests that deregulated RNA splicing factors play critical roles in tumorigenesis; however, their specific involvement in colon cancer remains largely unknown. Here we report that the splicing factor RBM25 is overexpressed in colon cancer, and this increased expression correlates with a poor prognosis of patients with colon cancer. Functionally, RBM25 ablation suppresses the growth of colon cancer cells both in vitro and in vivo. Mechanistically, our transcriptome-wide analysis of splicing events revealed that RBM25 regulates a large number of cancer-related alternative splicing events across the human genome in colon cancer. Particularly, RBM25 regulates the splicing of MNK2 by interacting with the poly G rich region in exon 14a, thereby inhibiting the selection of the proximal 3 ' splice site (ss), resulting in the production of the oncogenic short isoform, MNK2b. Knockdown of RBM25 leads to an increase in the MNK2a isoform and a decrease in the MNK2b isoform. Importantly, re-expression of MNK2b or blocking the 3 ' ss of the alternative exon 14a with ASO partially reverses the RBM25 knockdown mediated tumor suppression. Moreover, MNK2b levels were significantly increased in colon cancer tissues, which is positively correlated with the expression level of RBM25. Collectively, our findings uncover the critical role of RBM25 as a key splicing factor in colon cancer, suggesting its potential as a prognostic marker and therapeutic target.