Background and Aims Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 Ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2, and elevated RSK2 expression supporting oncogenic functions has been reported in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC. Methods We performed exome-sequencing and targeted DNA-sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential downstream pathways underlying RSK2 mutations were investigated by RNA-sequencing, qRT-PCR and mass-spectrometry. Results We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts, and revealed that HCC is the top-ranking cancer harboring RSK2 mutations among many cancer types. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring the RSK2 expression in the natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2 inactivating mutations alleviated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to Sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced the cholesterol biosynthesis-related gene expression in HCC cells. Conclusions Our findings unveil the mechanistic and functional significance of RSK2 inactivating mutations in HCC, and such inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC. Lay summary In this study, we identified and functionally characterized RSK2 inactivating mutations in human HCCs and demonstrated their association with aggressive tumor behavior. RSK2 mutation-driven MAPK activation could sensitize sorafenib treatment and activates cholesterol biosynthesis.