Hepatocellular carcinoma (HCC) is one of the malignant and lethal cancers. Single nucleotide polymorphisms (SNPs) in microRNAs(miRNAs) can affect the expression and target identification of miRNAs and lead to the formation of malignant tumors. However, little is known about whether microRNA-1269a (miR-1269a) SNPs affect the susceptibility and progression of HCC or their specific mechanism. The association between microRNA-1269a rs73239138 and the susceptibility to HCC was verified by MassARRAY assay in a large case-control sample. The effect of miR-1269a and the variant on the proliferation and apoptosis of HCC cells was examined by flow cytometry (FCM), CCK8 assay and Western blot. The target of miR-1269a was identified by bioinformatics analysis and qRT-PCR and its role on cell proliferative capacity was examined by CCK8 assay. The expression level of miR-1269a was analyzed by qRT-PCR in HCC cells transfected with wild or variant type pre-miR-1269a plasmid. MiR-1269a produced a tumor suppressor effect by inhibiting cell proliferation and inducing apoptosis of human HCC cells, possibly via inhibiting the expression of its target genes SPATS2L and LRP6, which were tumor promoters. While, rs73239138 (G >A) in miR-1269a reduced the anticancer effect of miR-1269a possibly by attenuating its total amount in HCC cells or its target recognition, reduce its inhibition on target genes and promoted the susceptibility to HCC. Our findings for the first time proved that miR-1269a SNP plays a role in the occurrence and process of HCC and the relevant mechanism, in accompany with the discovery of the novel target genes of miR-1269a.