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Recently, we demonstrated that two members of neurotrophins, nerve growth factor and brain-derived neurotrophic factor, and two types of receptor, tyrosine kinase A (TrkA) and tyrosine kinase (TrkB), exist in ejaculated bull spermatozoa, and play a crucial role in the normal function of spermatozoa. Neurotrophin-4 (NT-4) is another neurotrophic factor that signals predominantly through the TrkB receptor tyrosine kinase, and no reports of detection of NT-4 in spermatozoa have been published. In the present study, the presence of NT-4 in mature bull spermatozoa was investigated using RT-PCR, immunofluorescence and Western blotting. The result shows that there was no RT-PCR evidence for NT-4 transcripts in bovine spermatozoa. However, the NT-4 protein was present in bovine spermatozoa, and the NT-4 immunoreactivity was localized to the equatorial segment and midpiece of bovine spermatozoa. In addition, effects of NT-4 on function of spermatozoa were studied. Significant increased mitochondria activity of mature bovine spermatozoa was observed in response to 300 or 500 ng/ml exogenous NT-4 (p < 0.05), in comparison with the control, while addition of inhibitors (40 ng/ml k252α) specific for tyrosine protein kinase significantly blocked the increase of mitochondria activity. However, NT-4 had no effects on the viability or acrosome reaction of spermatozoa (p > 0.05). Consequently, this study provided evidence that NT-4 protein was presented in the mature bull spermatozoa and can influence the mitochondrial activity of bovine spermatozoa through TrkB tyrosine kinase–dependent pathways.