Multi-walled carbon nanotubes (MWCNTs) have been widely used in many fields and were reported to cause reversible testis damage in mice at high-dose. However the reproductive effects of low dose MWCNTs remained elusive. Herein, we used the mice spermatocyte cell line (GC-2spd) to assess the reproductive effects of MWCNTs. Size distribution, zeta potential, and intensity of MWCNTs were characterized. A maximal concentration of 0.5 μg/mL MWCNTs was found to be nonlethal to GC-2spd. At this dose, cell cycles and the ROS levels were in normal status. We also found MWCNTs accumulated in mitochondria, which caused potential mitochondrial DNA damage in spermatocyte. Furthermore, the expression level of mitochondria-related genes, the oxygen consumption rate, and cellular ATP content were declined compared to controls, even at the nonlethal dose. Our results suggested for the first time that, in germ cells, mitochondrion was a cellular organelle that accumulated MWCNTs.