Arsenic trioxide (As2O3; ATO), a compound which is characterized by its ability to function as a potent anticancer agent, has been investigated in a variety of carcinomas. B7‑H4, a transmembrane protein, may inhibit the function of the T cell effector, and therefore, may be useful in investigating different types of tumor therapies. However, few studies have been published previously associated with the roles of ATO and B7‑H4 in human hepatocellular carcinoma (HCC). The aim of the present study was to investigate the anti‑invasive role of ATO in HCC, to determine the effect of ATO treatment on the expression of B7‑H4 and to further assess the possible underlying mechanisms. Following treatment of the cells with 2, 4 and 8 µM ATO for 48 h, cell counting kit‑8 (CCK‑8), Transwell and western blot assays were used to determine the extent of human MHCC97‑H HCC cell proliferation, apoptosis, invasion and B7‑H4 expression, respectively. The results revealed that 1 µM ATO markedly decreased cellular proliferation, and ATO administered at concentrations of 0.1, 0.2 and 0.5 µM markedly inhibited the migration and invasion of the human MHCC97‑H HCC cell line. The expression of B7‑H4 in the treatment groups was markedly reduced. Signal transduction mediated via the Janus kinase 2/signal transducers and activators of transcription 3 pathway was inhibited upon treatment with 0.1, 0.2 and 0.5 µM ATO. Additionally, the protein expression levels of matrix metalloproteinase 2 and vascular endothelial growth factor were markedly reduced in HCC cells upon treatment with ATO. In conclusion, ATO may reduce the protein expression levels of B7‑H4 in MHCC97‑H HCC cells, and further affected HCC tumorigenesis and progression. ATO may be a putative agent for the development of therapeutic strategies against human liver cancer.