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To analyze the mechanism of testis-specific protein Y-encoded 1 (TSPY1) in male hepatocellular carcinoma (HCC).This experimental study was carried out at Guangxi Medical University's First Affiliated Hospital, Guangxi, China, between January 2016 and December 2019. The expression of TSPY1, androgen receptor (AR), messenger ribonucleic acids (mRNAs), and proteins were detected by qRT-PCR and Western blotting. The co-localization and interaction of TSPY1 and AR were observed by immunofluorescence assay and co-immunoprecipitation. Hepatocellular carcinoma cells overexpressing and silencing TSPY1 were constructed, and the expression and phosphorylation levels of TSPY1, AR, and mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway-related key molecules ERK1/2, p38, and JNK were also detected.The expression levels of TSPY1, AR mRNAs, and proteins were highly positively correlated in HCC cells in different metastatic potentials with a high correlation coefficient of R=0.929 and R=0.884. Testis-specific protein Y-encoded 1 and AR were then co-localized in the nucleus of HCC cells, and TSPY1 and AR can interact with each other. In addition, the expression of AR and phosphorylation of ERK1/2 were enhanced in TSPY1 overexpressed Huh7 cells. They were reduced in HCCLM3 cells with TSPY1 knockdown expression. In addition, in response to blocking MAPK/ERK signaling activity, AR was reduced in expression.These findings suggested that there was a positive correlation between TSPY1 expression and AR in male HCC cells, and high TSPY1 expression stimulates AR expression, MAPK/ERK signaling pathway may be involved in its mechanism.