Abstract Background and Purpose: Indisulam, a synthetic sulfonamides drug, has been reported to harbor anticancer activity in multiple tumor models, especially in colorectal cancer, lung cancer and Glioblastoma. However, the efficacy of indisulam has still not been tested in human cervical carcinoma cells. Experimental Approach: The human cervical carcinoma HeLa cell lines were treated with different concentration of indisulam. Cell viability, cell cycle and apoptosis analysis, Western blot, RNA-seq sequencing, bioinformatics analysis and immunofluorescent staining were performed. Key Results: We found that indisulam is highly efficacious against HeLa cells, leading to significantly reduced cell growth and post-translational RBM39 protein degradation in a dose dependent manner. RNAseq and proteomics analysis also highlighted a disruption to transcription, mRNA splicing, cells cycle arrest and apoptotic process related pathways. In addition, treatment of HeLa cells with indisulam lead to G1/S phase arrest by inhibiting expression of CDK2 and Cyclin E1. High concentration of indisulam treatment can induce pro-apoptotic p73 isomers splicing and subsequent cell apoptosis of mitochondrial pathway. Conclusion and Implications: Our data suggests that indisulam has therapeutic potential in cervical cancer, representing an attractive strategy that should be further investigated.