Hepatocellular carcinoma (HCC) is the fifth-most common cancer and third leading cause of cancer-related deaths worldwide. Increasing evidence indicates that dysregulation of microRNAs is often observed in HCC, and has been extensively investigated in terms of cancer formation, progression, diagnosis, therapy, and prognosis. Recently, microRNA-326 (miR-326) has been demonstrated to play important roles in multiple types of human cancer. However, the expression pattern, clinical significance, roles and regulatory mechanisms of miR-326 in HCC have yet to be elucidated. In this study, miR-326 was frequently downregulated in HCC tissues and cell lines. Low miR-326 expression was significantly associated with the TNM stage, differentiation and lymph node metastasis of HCC patients. Further functional assays demonstrated that the recovered miR-326 expression inhibited HCC cell proliferation and invasion and activated cell apoptosis in vitro. In addition, LIM and SH3 protein 1 (LASP1) was identified as a direct target gene of miR-326 in HCC. Furthermore, LASP1 was upregulated in HCC tissues and cell lines. The expression level of LASP1 mRNA was inversely correlated with that of miR-326 in HCC tissues. Moreover, LASP1 silencing elicited effects similar to miR-326 overexpression on HCC cells, and LASP1 upregulation markedly reversed the effects of miR-326 overexpression on HCC cells. These results revealed that miR-326 suppressed the progression of HCC by directly targeting LASP1. Therefore, miR-326 may be used as a potential therapeutic target for the treatment of patients with HCC.