Background: Prostate cancer (PC) is common among the elderly and significantly impacts their quality of life. This study aims to explore the role of acetyl-CoA acetyltransferase 1 (ACAT1) in the cellular process of PC cells and the interplay of ACAT1 and the Wnt/beta-catenin pathway.Methods: Initially, we assessed the expression level of ACAT1 in normal and PC cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. Subsequently, after transfection with ACAT1 overexpression plasmid or siACAT1, we conducted a series of functional assays, including cell counting kit-8 (CCK-8), Transwell and flow cytometry, to evaluate the biological behaviors of PC-3 cells. qRT-PCR and Western blot were selected to determine expressions of apoptosis-related proteins, Wnt/beta-catenin and its downstream genes. Later, PC-3 cells received treatment with FH535 or LiCl and transfection with ACAT1 overexpression plasmid/siACAT1, after which the cell's biological behaviors were determined again.Results: ACAT1 was expressed more in PC cells than in RWPE-1 cells (p < 0.001). Overexpressed ACAT1 exerted the effects of potentiating cell proliferation, migration and invasion and suppressing cell apoptosis (p < 0.001). Also, under the inducement of overexpressed ACAT1, levels of B cell lymphoma-2 (Bcl-2) and Wnt/beta-catenin signaling and its downstream genes were elevated, while those of Bcl-2-associated X protein (Bax) and cleaved caspase-3 were repressed (p < 0.001). However, ACAT1 knockdown produced inverse results (p < 0.001). FH535 treatment repressed ACAT1 overexpression-induced proliferation, migration and invasion, while LiCl treatment reversed the negative effects of ACAT1 silencing (p < 0.001).Conclusions: ACAT1 enhances the biological functions of PC cells by modulating the Wnt/beta-catenin signaling pathway and its downstream genes.