AbstractNumerous recent studies suggest that cancer‐specific splicing alteration is a critical contributor to the pathogenesis of cancer. RNA‐binding protein with serine‐rich domain 1, RNPS1, is an essential regulator of the splicing process. However, the defined role of RNPS1 in tumorigenesis still remains elusive. We report here that the expression of RNPS1 is higher in cervical carcinoma samples from The Cancer Genome Atlas (TCGA‐cervical squamous cell carcinoma and endocervical adenocarcinoma) compared to the normal tissues. Consistently, the expression of RNPS1 was high in cervical cancer cells compared to a normal cell line. This study shows for the first time that RNPS1 promotes cell proliferation and colony‐forming ability of cervical cancer cells. Importantly, RNPS1 positively regulates migration‐invasion of cervical cancer cells. Intriguingly, depletion of RNPS1 increases the chemosensitivity against the chemotherapeutic drug doxorubicin in cervical cancer cells. Further, we characterized the genome‐wide isoform switching stimulated by RNPS1 in cervical cancer cell. Mechanistically, RNA‐sequencing analysis showed that RNPS1 regulates the generation of tumor‐associated isoforms of key genes, particularly Rac1b, RhoA, MDM4, and WDR1, through alternative splicing. RNPS1 regulates the splicing of Rac1 pre‐mRNA via a specific alternative splicing switch and promotes the formation of its tumorigenic splice variant, Rac1b. While the transcriptional regulation of RhoA has been well studied, the role of alternative splicing in RhoA upregulation in cancer cells is largely unknown. Here, we have shown that the knockdown of RNPS1 in cervical cancer cells leads to the skipping of exons encoding the RAS domain of RhoA, consequently causing decreased expression of RhoA. Collectively, we conclude that the gain of RNPS1 expression may be associated with tumor progression in cervical carcinoma. RNPS1‐mediated alternative splicing favors an active Rac1b/RhoA signaling axis that could contribute to cervical cancer cell invasion and metastasis. Thus, our work unveils a novel role of RNPS1 in the development of cervical cancer.