Background: Direct-acting antivirals (DAAs) have achieved a sustained virological response (SVR) rate of 95-99% in treating HCV. Several studies suggested that treatment with sofosbuvir (SOF) containing DAAs may be associated with increased risk of developing HCC. The aim of this study is to investigate the potential mechanisms of SOF on the development of HCC.Methods: This study used OR-6 (harboring full-length genotype 1b HCV) and Huh 7.5.1 cells to examine the effects of SOF on cell proliferation and migration in HCC cells. SOF-upregulated genes in OR-6 cells were inspected using next generation sequencing (NGS) and the clinical significance of these candidate genes was analyzed using The Cancer Genome Atlas (TCGA) database. siRNA-mediated knockdown of candidate genes and their influence on cell proliferation and migration were also examined.Results: SOF increased cell proliferation and cell migration in OR-6 and Huh7.5.1 cells in both two- and three-dimensional culture models. NGS identified several genes that were significantly upregulated by SOF in OR-6 cells. Among these genes, PHOSPHO2, KLHL23, TRIM39, TSNAX-DISC1 and RPP21 expression were significantly elevated in the tumor part compared with the non-tumor part of HCC in the TCGA database. PHOSPHO2 and RPP21 were found to be associated with overall survival of HCC patients in the TCGA database. Moreover, knockdown of PHOSPHO2-KLHL23, TSNAX-DISC1, TRIM39 and RPP21 ameliorated cell proliferation and migration increased by SOF in OR-6 and Huh 7.5.1 cells. Conclusion: SOF upregulated several genes that were associated with increased cell proliferation and migration and the development of HCC.