Hepatocellular carcinoma is a common type of liver cancer. Resistance to chemotherapeutic agents is a major problem in cancer therapy. MicroRNAs have been reported in cancer development and tumor growth; however, the relationship between chemoresistance and hepatocellular carcinoma needs to be fully investigated. Here, we treated hepatocellular carcinoma cell line (HA22T) with a histone deacetylase inhibitor to establish hepatocellular carcinoma-resistant cells (HDACi-R) and investigated the molecular mechanisms of chemoresistance in HCC cells. Although histone deacetylase inhibitor could not enhance cell death in HDACi-R but upregulation of miR-107 decreased cell viability both in parental cells and resistance cells, decreased the expression of cofilin-1, enhanced ROS-induced cell apoptosis, and dose-dependently sensitized HDACi-R to HDACi. Further, miR-107 upregulation resulted in tumor cell disorganization in both HA22T and HDACi-R in a mice xenograft model. Our findings demonstrated that miR-107 downregulation leads to hepatocellular carcinoma cell resistance in HDACi via a cofilin-1-dependent molecular mechanism and ROS accumulation.