In both humans and animals, approximately half of all cases of infertility derived from male factor or sub-fertility. However, the molecular mechanisms of sperm fertility defects are not well known. Coupled with the fact that the mature spermatozoa have lost the transcription, translation, and protein synthesis, proteomic analysis is acceptable to understand comprehensive process of sperm molecular function related with fertility. The recent developments in proteomic study have amplified the identification of biomarkers associated with fertility. Despite these recent efforts, no studies have clearly defined the relationship between the proteome and male fertility after proteomic study. Therefore, we used proteomic approaches to study fertility-related marker expression in the spermatozoa of high and low fertility bulls, and we used western blot analysis to evaluate whether the proteins identified by 2-DE were correlated with the fertility of 20 individual bovine spermatozoa. We identified eight proteins that were differentially expressed in the spermatozoa of high and low fertility bulls. Five proteins, ENO1, ATP5B, ASPP2, AHSG, and GPX4, were more highly represented in the spermatozoa of high fertility bulls; whereas three proteins, VDAC2, ropporin-1, and UQCRC2, were more highly represented in the spermatozoa of low fertility bulls. The expression levels of ENO1 were greater in the spermatozoa of high fertility bulls than low fertility bulls (Figure 2 A), and ENO1 expression was strongly positively correlated with NRR (r = 0.517, p < 0.05, Figure 3 A). There were no differences in protein expression between high and low fertility bulls for ATP5B and PHGPx4 (Figure 2 B and Figure 2 C), and the expression levels of these proteins were not significantly correlated with NRR (ATP5B: r = 0.1, PHGPx4: r = 0.053, both p < 0.05; Figure 3 B and Figure 3 C). The expression levels of UQCRC2 and VDAC2 were significantly negatively correlated with historic NRR (UQCRC2: r = −0.503, p < 0.05, Figure 2 D; VDAC2: r = 0.475, p < 0.05, Figure 2 E). To my best knowledge, we suggested that concurrent comparisons between protein expression and male fertility by western blot analysis may represent a good in vitro model system to diagnosis the male fertility. Moreover, these novel proteins will used to not only major male fertility markers and further explore the mechanism of male fertility, but will also provide new targets for male contraceptives. This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ008415), Rural Development Administration, Republic of Korea.