Objective: Explore the effect of GATA5 expression on Paclitaxel inhibiting growth of hepatocellular carcinoma (HCC) cells. Materials and Methods: In the experimental study, HCC cell lines (HLE, Bel7402 and PLC/PRF/5) were treated with different concentrations of Paclitaxel (5-20 mg/ml) for 24 hours. HLE cells were transfected with GATA5-siRNA vector, while Bel7402 and PLC/PRF/5 cells were transfected with overexpressed GATA5 vector for 24 hours, followed by treatment of the cells with Paclitaxel (10 mg/ml) for 24 hours and subsequently 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay to detect growth of HCC cells. Soft agar cultured was used to analyze formation of colony. Apoptosis of HCC cells were detected by Flow cytometer. Migration of HCC cells was observed by trawell assays. Western blotting and laser confocal microscopy were utilized to detect expression and location of the proteins. Results: Inhibiting expression of GATA5 reduced sensitivity of HLE cells to Paclitaxel, while overexpression of GATA5 increased sensitivity of Bel7402 cells and PLC/PRF/5 cells to Paclitaxel. Overexpression of GATA5 played a role in stimulating Paclitaxel to inhibit growth, colony formation and migration, as well as enhance apoptosis in HCC cells. Overexpression of GATA5 also promoted Paclitaxel to inhibit expression of reprogramming genes, such as Nanog, EpCAM, c-Myc and Sox2 in Bel7402 and PLC/PRF/5 cells. Inhibited expression of GATA5 led to enhancement of the expression of CD44 and CD133, in HLE cells. Overexpression of GATA5 was not only alone but also synergized with Paclitaxel to inhibit expression of CD44 and CD133 in Bel7402 or PLC/PRF/5 cells. Conclusion: Overexpression of GATA5 played a role in enhancing Paclitaxel to inhibit the malignant behaviors of HCC cells. It was involved in suppressing expression of the reprogramming genes and stemness markers. Targeting GATA5 is an available strategy for applying paclitaxel to therapy of patients with HCC.