This study was designed to evaluate the effect of fluoride (F) on the cell cycle, apoptosis, and DNA damage in TM3 mouse Leydig cells in vitro by the methods of flow cytometry (FCM) and single cell gel electrophoresis (SCGE). TM3 mouse Leydig cells were exposed to 0, 200, 400, 600, and 800 umol NaF/L for 24 hr. By FCM, the number of cells in G0/G1 phase were much higher, whereas the cells in S phase, G2+M phase, and the proliferation index (PI) value were much lower than in the control group. Moreover, the percentage of Leydig cell apoptosis increased with increasing concentration of F compared with the control group. From SCGE, tail length increased with increasing concentration of F, and the effects were dose-dependent. The results show that F can cause cell cycle aberration, induce apoptosis, and DNA damage in cultures of TM3 mouse Leydig cells.