Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced by Fusarium species. The exposure risk to humans and animals is the consumption of contaminated food and animal feeds. The aim of this study was to investigate ZEA-induced effects and its tumorigenic mechanism in TM3 cells (mouse Leydig cells). Cell proliferation, apoptosis, and gap junction intercellular communication (GJIC) were assessed in this study. Results showed that low concentrations of ZEA could significantly promote the growth of TM3 cells. The percentage of cell distribution was decreased significantly in G1/G0 phase and was increased significantly in S phase with 10 and 20 μg/L of ZEA for 72 h (p < 0.05, p < 0.01). The expressions of cyclin D1 and Cdk4 were significantly increased in the exposure groups compared with the control group (p < 0.05, p < 0.01). Compared with the control group, the apoptosis was significantly decreased in 10 and 20 μg/L groups (p < 0.01), and the ratio of Bax/Bcl-2 protein level was significantly decreased in a dose-dependent manner. The protein levels of proto-oncogene c-Myc, c-Jun, and c-Fos were significantly elevated and the protein levels of anti-oncogene p53 and phosphatase and tensin homolog (PTEN) were decreased obviously compared with the control group (p < 0.05, p < 0.01). ZEA affected the expressions of connexins and inhibited the activity of GJIC. These results demonstrated that ZEA can disturb the dynamic balance between proliferation and apoptosis and causes abnormal regulation of oncogenes, GJIC, and connexins in TM3 cells, which may easily induce the translation of normal cells into tumor cells.