We have investigated the expression and role of the 58-kDa micro-spherule protein (MSP58) in hepatocellular carcinoma (HCC). Immunohistochemistry was performed in 252 samples from patients with HCC to detect the expression level of MSP58. Results indicated that the expression level of MSP58 in the cancer samples was significantly higher than that in adjacent normal tissues. The Wilcoxon-Mann-Whitney test showed significant difference in the expression of MSP58 in patients with serum AFP, tumor size, histological differentiation, and universal integrated circuit card (UICC) stage (P < 0.001, P = 0.004, P < 0.001, P < 0.001, respectively). A total of 252 HCC patients were followed up for five consecutive years, and Kaplan-Meier survival analysis demonstrated that the survival time of HCC patients with low expression of MSP58 was longer than those with high expression during the 5-year follow-up period (P < 0.001). Cox regression analysis indicated that high expression of MSP58 (++ or +++), serum AFP (≥25 μg/L), tumor size (≥3 cm), and UICC stage (III or IV) were the independent poor prognostic factors of HCC (P = 0.008, 0.0290, 0.001, 0.047, respectively). Furthermore, down-regulation of MSP58 was introduced to HCC cell lines (HepG2 and Huh7) by plasmid transfection. In vivo and in vitro studies indicated that MSP58si markedly reduced proliferation and promoted the apoptosis of HepG2 and Huh7 cells. In summary, our results demonstrated that MSP58 played an important role in the proliferation and apoptosis of HCC cells and the expression of MSP58 in HCC patients was closely related to the prognosis.