Objective To investigate the inhibitory effect of arsenic trioxide (ATO) on the expression of peptidylprolyl cis-trans isomerase NIMA-interacting 1 (Pin1) in liver cancer and its molecular mechanism. Methods The expression of Pin1 in human hepatocellular carcinoma cell lines and liver cancer tissue was analyzed using DepMap database and GEPIA database. Human hepatocellular carcinoma cell line Huh7 and mouse hepatocellular carcinoma cell line H22 were used as cell models to detect the effect of ATO on tumor cell viability by adenosine triphosphate method. The effects of ATO on Pin1 expression at protein and transcription levels were detected by Western blotting, immunofluorescence staining and quantitative polymerase chain reaction (qPCR). After pretreatment with chloroquine to inhibit lysosome pathway in Huh7 cells, the regulation effect of ATO on Pin1 expression was detected by Western blotting and immunofluorescence staining. The effects of ATO on hepatoma cell growth and Pin1 expression in vivo were verified by subcutaneous tumor-bearing mouse model and immunohistochemical staining. The possible signaling pathways affected by ATO were analyzed by RNA sequencing and were verified by Western blotting and qPCR. Results The expression of Pin1 in 23 kinds of human hepatocellular carcinoma cell lines in DepMap database and human hepatocellular carcinoma tissue in GEPIA database showed a high level. In vitro, the viability of Huh7 and H22 cells was decreased after ATO treatment, and the protein and transcription levels of Pin1 were decreased. The effect of ATO on Pin1 expression was reversed after inhibiting lysosomal pathway by chloroquine. In the subcutaneous tumor-bearing mouse model, ATO showed certain anti-tumor effects, and immunohistochemical staining showed that Pin1 expression and tumor cell proliferation were inhibited after ATO treatment. The genes related to Wnt/β-catenin pathway were enriched in ATO-treated H22 cells, and the expression of β-catenin was decreased after inhibiting Pin1 expression in H22 cells. Conclusion ATO inhibits proliferation of hepatocellular carcinoma cells by inhibiting Pin1 expression through lysosomal pathway and affecting Wnt/β-catenin signaling pathway.