Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that underwent alternative splicing (AS) and generated a spliced isoform in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with pre-mRNA located at the region around the 3'- splicing site on intron 2 and the alternative 3'- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of , and exogenous expression of enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating splicing induced by IR, providing potentially effective radiotherapy by modulating splicing against HCC.