Human Ataxin-3 protein was first identified as a transcript from patients with Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3). Recent studies have demonstrated that Ataxin-3 is involved in gastric cancer and lung cancer. However, the role of Ataxin-3 in testicular cancer (TC) remains poorly understood. This study aims to explore the significance of Ataxin-3 expression in TC. Firstly, we investigated 53 paired TC and para-tumor tissues and found that Ataxin-3 was overexpressed in TC tissues, and this overexpression of Ataxin-3 was correlated with tumor stages. Functionally, Ataxin-3 overexpression promoted cell proliferation, and Ataxin-3 knockdown inhibited cell proliferation. In addition, up-regulation of Ataxin-3 inhibited the expression of PTEN and activated the AKT/mTOR pathway. Conversely, inhibition of Ataxin-3 suppressed the expression of p-AKT and p-mTOR, and increased the expression of p-4EBP1. These findings may provide a better understanding about the mechanism of TC and suggest that Ataxin-3 may be a potential prognostic biomarker and therapeutic target for TC.