Updated project metadata. Abnormal alternative splicing (AS) caused by alterations to splicing factors contributes to tumor progression. Nonetheless, the relevant targets and mechanisms remain elusive in hepatocellular carcinoma (HCC). Here, we reported that overexpression of Ubiquitin-specific protease 39 (USP39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is associated with poor clinical outcomes and proliferative signaling. Functionally, hepatocyte-specific USP39 knockin mice exhibited enhanced hepatocarcinogenesis. In vitro, USP39 promoted HCC cell proliferation and cell cycle progression in a spliceosome-dependent manner. Transcriptomic analysis revealed that USP39 depletion led to comprehensively impaired constitutive splicing and intriguingly, selective AS of hundreds of genes. USP39-mediated splicing switch of KANK2-S to KANK2-L increased the tumorigenic potential of HCC cells through accelerating KANK2 translation. Mechanistically, USP39 modulates exon inclusion/exclusion via interaction with SRSF6 or hnRNPC in a position-dependent manner. These findings highlight a role for USP39 as a splicing regulator in HCC biology and establishing its position-dependent splicing model.