Bombyx mori was used to study the molecular mechanism of fluoride induced reproductive toxicity. In our previous study, we confirmed the physiological and biochemical effects of NaF on reproductive toxicity, and we found that the molecular mechanism of NaF induced reproductive damage may be associated with the oxidative phosphorylation pathway. To further study the function of NaF exposure on the oxidative phosphorylation pathway in the testis in Bombyx mori, and the relationship between oxidative phosphorylation and oxidative stress, we measured the changes in the main ROS (O2- and H2O2) in the testis, the activity of the main electron transport chain complex enzymes in the oxidative phosphorylation pathway and the transcription levels of the corresponding genes; we additionally performed pathological observations of the silkworm testis after exposure to 200 mg/L NaF solution for different times. The content of O2- and H2O in the silkworm gonads increased significantly at 24 h, 72 h and 120 h after NaF stress. The activity of mitochondrial complexes I, III, IV and V in the silkworm testis was significantly greater than that in the control group. RT-PCR analysis suggested that the mRNA transcription levels of NADH-CoQ1, Cyt c reductase, Cyt c oxidase and ATP synthase genes were up-regulated significantly. Histopathological investigation showed that the damage to the silkworm testis was more severe with increasing NaF exposure times. These results indicated that NaF stress affects the NADH respiratory chain of the mitochondrial electron transport chain and increases the activity of related enzyme complexes, thus destroying the balance of the electron transport chain. Subsequently, the content of ROS in cells significantly increases, thus resulting in oxidative stress reactions in cells. These results enable better understanding of the testis-damaging molecular toxicological mechanism of NaF.