Motorcycles are one of the primary means of transportation in many Asian metropolitan areas. Exposure to motorcycle exhaust (ME) has been shown to cause male reproductive toxicity in a rat inhalation model. In the present study, the molecular mechanisms underlying ME-induced male reproductive toxicity were examined in a rat model using nuclear magnetic resonance (NMR)-based metabolomics. Rats were treated with 1:10-diluted ME for 1 hour in the morning and 1 hour in the afternoon, daily, via head-nose-only exposure chambers, for a period of four weeks. Half of the ME-exposed rats were co-treated with vitamin E to examine its protective effects. Both hydrophilic and hydrophobic metabolites from the testes and liver were extracted for 1H and J-resolved NMR analyses. The obtained NMR spectra were converted into series of spectral binned areas and subjected to orthogonal projections to latent structure-discriminant analyses. Decreased spermatid counts and elevated levels of betaine in the testes of rats in the ME-treated group were partially attenuated by vitamin E co-treatment. Although it was not statistically significant, we observed a consistent trend of partial reversal of the elevated levels of alanine, glycine, leucine, tyrosine, and valine in the testes of rats in the ME-treated group due to vitamin E co-treatment. Decreases in testicular ethanolamine and inositol were only significant in the group co-treated with ME and vitamin E. The testicular toxicity of ME may be due to alterations in lipid- and energy-related metabolism. We conclude that ME generates greater metabolic responses in the testes than in the liver. Treatment with vitamin E can partially reverse the molecular events in the testes.