Hepatocellular carcinoma (HCC) is the third highest cause of cancer-related deaths globally. One of the cellular hallmarks of this disease is dysregulation of apoptosis, and a better understanding of this process is important if progress is to be made toward effectively treating HCC. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a RNA-binding protein that is implicated in apoptosis and is upregulated in various cancers, including HCC. In this study, we report new evidence for a crucial role of hnRNP K in suppressing apoptosis in HCC cells. We used the chemotherapeutic agent 5-fluorouracil to induce apoptosis in HCC cell lines and found that hnRNP K was downregulated, independent of both p53 and caspases. Prolonged downregulation of hnRNP K using small interfering RNA (siRNA) significantly decreased cell viability and increased apoptosis in HCC cell lines in a p53-independent manner. Moreover, enhanced tumor necrosis factor-related apoptosis-inducing ligand potency, independent of BH3-interacting domain death agonist (BID) cleavage, was also observed in hnRNP K siRNA-treated cells. Examination of the underlying mechanism revealed that hnRNP K suppresses the activity of various caspases through controlling transcription of the caspase inhibitor XIAP. Taken together, this study establishes that hnRNP K plays an antiapoptotic role in HCC cell lines, independent of p53 status, via the maintenance of high levels of endogenous caspase inhibitors, and also identifies hnRNP K as a possible therapeutic marker for cancer treatment.