Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy, and multiple host genetic factors are believed to contribute to HCC development. The small G protein signaling modulator 3 (SGSM3) has been shown to be associated with small G protein coupled receptor signal transduction pathway, suggesting a potential role in HCC susceptibility. We carried out a case-control study in a Chinese population (502 cases and 513 controls) to determine whether the 4-bp insertion/deletion polymorphism (rs56228771) in 3' untranslated region of SGSM3 could affect HCC susceptibility. Logistic regression analysis showed that compared with the del/del genotype, the ins/del genotype of rs56228771 was associated with a significantly decreased risk of HCC [adjusted odd ratio = 0.55, 95% confidence interval (CI) = 0.42-0.73, P = 1.93 × 10(-5)]. The combined ins/del + ins/ins genotypes contributed to a 45% decreased HCC risk (95% CI = 0.42-0.73, P = 1.03 × 10(-5)). This protective trend was more prominent in the HBsAg-negative subgroup. Furthermore, in vivo experiments showed that mRNA levels of SGSM3 from HCC tumor tissues and adjacent non-HCC tissues were correlated with rs56228771 genotypes. Tissue samples with ins/ins genotype have the highest level of SGSM3, which are 2.85-3.00-fold and 1.46-1.57-fold higher than that with ins/del and del/del genotype, respectively. Similar results were also observed with four common hepatoma cell lines in vitro. In addition, compared with HCC tissues, significantly higher SGSM3 expression was observed in adjacent non-HCC tissues (fold change = 2.48), implying its tumor suppressor roles in HCC. Bioinformatics prediction showed that the insertion allele disrupted a binding site for microRNA (miRNA)-151-5p, which would upregulate SGSM3. Taken together, we provided initial evidence that rs56228771 may contribute to hepatocarcinogenesis, possibly by affecting SGSM3 expression through a miRNA-mediated regulation. The replication of our studies in other populations and functional analysis will further strengthen the underlining mechanism.