Objective To study the expression of Musashi-1 in tumor tissues and adjacent non-tumor tissues of patients with hepatocellular carcinoma (HCC), and to explore the relationship between Musashi-1 and clinical prognosis and the effect of Musashi-1 on the metastasis and invasion of HCC cells. Methods We collected HCC tissues and adjacent non-tumor tissues from 138 patients undergoing radical resection of HCC in our hospital from June 2000 to June 2010. The expression of Musashi-1 in all tissues was detected by immunohistochemical method. The relationship between Musashi-1 expression and clinical characteristics and prognosis was analyzed by Pearson χ² test and Fisher exact test. The expression of CD133 in HCC tissues and adjacent non-tumor tissues was detected by qPCR and the correlation between Musashi-1 and CD133 in tumor tissues was analyzed. Finally, we constructed SMMC-7721 cells with stable overexpression of Musashi-1, determined the effect of Musashi-1 on the sphere-forming potential of SMMC-7721 cells using Sphere formation assay, and detected the effect of Musashi-1 on the ability of cell invasion by tumor invasion experiment. Results Musashi-1 was highly expressed in 105 (76.1%) HCC tissues compared with the adjacent non-tumor tissues (P<0.05). The expression of Musashi-1 was related to α-fetoprotein (AFP) level, ascites and distant metastasis (all P<0.05), but not to gender, age, HBV contagious history, tumor size, tumor foci, portal vein tumor thrombus or microscopic tumor thrombus (P>0.05). Spearman analysis indicated that HCC patients with higher Musashi-1 expression have significantly worse overall survival rate (P<0.05) and disease-free survival (P<0.05) compared with those with low Musashi-1 expression. The expression of CD133 mRNA in HCC tissues was significantly higher than that in the adjacent non-tumor tissues (P<0.05), and CD133 expression was positively correlated with Musashi-1 expression (R²=0.78, P<0.001). In vitro experiments confirmed that overexpression of Musashi-1 enhanced the sphere-forming potential and invasive ability of SMMC-7721 cells. Conclusion Musashi-1 has higher expression in HCC tissues and may promote HCC progression and metastasis. High level of Musashi-1 may indicate poor prognosis, providing a reference for prognosis judgment in HCC patients.