Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. And due to the low early diagnostic rate of liver cancer, many patients miss the optimal time for surgical resection, so it is necessitate to identificate the novel therapeutic targets. This study investigates the role of microRNA-6069 (miR-6069) in HCC pathogenesis. We analyzed miR-6069 expression in the TCGA-LIHC cohort, revealing significant upregulation in tumor tissues compared to adjacent normal tissues, and verified it in human tissues. MiR-6069 antisense oligonucleotide(ASO) effectively inhibits HCC cell proliferation in vitro and suppresses subcutaneous HCC tumor growth in nude mice without affecting their weight. Through bioinformatics analysis and immunohistochemistry, we identified PLEKHO1 as a target of miR-6069, and its expression is negatively correlated with miR-6069 expression. Furthermore, using immunohistochemical staining, quantitative PCR (QT-PCR), and Western blot (WB) analysis, we observed that the expression of PLEKHO1 significantly increased in the tumors of nude mice following miR-6069 ASO intervention, and affecting the expression of downstream molecules in the AKT/PI3K signaling pathway. These findings suggest that miR-6069 may influence HCC proliferation by modulating the AKT/PI3K signaling pathway.These findings highlight miR-6069 as a promising therapeutic target in HCC management.