Background: Brain-derived neurotrophic factor (BDNF) and its receptor, neurotrophic tyrosine receptor kinase-2 (NTRK2), are well known for their roles in the central nervous and animal reproductive systems. Several studies have observed the extensive expression of BDNF and NTRK2 in non-neuronal tissues, especially reproductive organs. However, most of these studies focused on ovarian development and regulation; thus, scientific research on BDNF and NTRK2 in males is required to determine their roles in the male reproductive system. Therefore, this study aimed to investigate BDNF and NTRK2 expression in bovine testes. Methods: Testes were collected from six Hanwoo bulls (6-8 months old). Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to investigate the mRNA expression of BDNF and NTRK2 in the testes. Western blot analysis was performed to verify the cross-reactivity of BDNF and NTRK2 antibodies with bovine testicular tissues. Immunohistochemistry was conducted to determine BDNF and NTRK2 protein expression in the testes. Results: RT-PCR analysis revealed BDNF and NTRK2 mRNA expression in bovine testes. In Western blotting, BDNF and NTRK2 protein bands were observed at 32 and 45 kDa, respectively. Immunofluorescence demonstrated BDNF expression in the nuclei of spermatogonia and Sertoli cells as well as in the cytoplasm of Leydig cells. NTRK2 was exclusively expressed in Sertoli cells. These results suggest that BDNF plays a potential role in spermatogenesis via BDNF and NTRK2 signaling in bovine testes, a finding supported by previous results in different animal species. Conclusions: The expression patterns of BDNF and NTRK2 indicate their functional importance in the bovine reproductive system.