This study analysed the microarray datasets from Gene Expression Omnibus (GEO) database, and aimed to identify novel potential hub genes associated with the progression of HCC via bioinformatics analysis and experimental validation. The common differentially expressed genes (DEGs) from five GEO datasets were screened using GEO2R tool. The expression and survival analysis of hub genes in HCC were performed using Gene Expression Profiling Interactive Analysis, UALCAN and Kaplan-Meier plotter tools. In vitro functional assays were used to determine the caspase-3, -9, cell proliferation and chemo-sensitivity of HCC cells. A total of 177 common DEGs were identified between normal liver and HCC tissues among these datasets. Functional enrichment and PPI network analysis identified 22 hub genes from the common DEGs. The mRNA expression of 22 hub genes was all significantly up-regulated in HCC tissues compared to that in normal liver tissues. Further survival analysis showed that 10 hub genes predicted poor prognosis of patients with HCC. More importantly, the in vitro functional studies demonstrated that KIF20A knockdown suppressed the HCC cell proliferation and promoted the chemosensitivity of HCC cells to cisplatin and sorafenib. In conclusion, the present study identified a total of 177 common DEGs among 5 GEO microarray datasets and found that 10 hub genes could predict the poor prognosis of patients with HCC using the comprehensive bioinformatics analysis. Furthermore, KIF20A silence suppressed cell proliferation and enhanced chemosensitivity in HCC cells. Further studies may be required to determine the mechanistic role of these hub genes in HCC progression.