Calf Spleen Extractive Injection (CSEI), a small peptides enriched extraction, performs immunomodulatory activity on cancer patients suffering from radiotherapy or chemotherapy. The present study aims to investigate the anti-hepatocellular carcinoma effects of CSEI in cells and tumor-xenografted mouse models. In HepG2 and SMMC-7721 cells, CSEI reduced cell viability, enhanced apoptosis rate, caused reactive oxygen species (ROS) accumulation, inhibited migration ability, and induced caspases cascade and mitochondrial membrane potential dissipation. CSEI significantly inhibited HepG2-xenografted tumor growth in nude mice. In cell and animal experiments, CSEI increased the activations of pro-apoptotic proteins including caspase 8, caspase 9 and caspase 3; meanwhile, it suppressed the expressions of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and anti-oxidation proteins, such as nuclear factor-erythroid 2 related factor 2 (Nrf2) and catalase (CAT). The enhanced phosphorylation of P38 and c-JunN-terminalkinase (JNK), and decreased phosphorylation of extra cellular signal-regulated protein kinase (ERKs) were observed in CSEI-treated cells and tumor tissues. CSEI-induced cell viability reduction was significantly attenuated by N-Acetyl-l-cysteine (a ROS inhibitor) pretreatment. All data demonstrated that the upregulated oxidative stress status and the altered mitogen-activated protein kinases (MAPKs) phosphorylation contributed to CSEI-driven mitochondrial dysfunction. Taken together, CSEI exactly induced apoptosis in human hepatocellular carcinoma cells via ROS/MAPKs dependent mitochondrial pathway.