Artificial insemination (AI) using cryopreserved sperm has an important positive impact on cattle production. Fertility is the most critical trait controlling livestock production; however, molecular, cellular, and physiological determinants of bull fertility and sperm freezability are not well understood. Better understanding of molecular, cellular, and physiological underpinnings of bull fertility may increase the success rate of AI. The objective of this study was to test the hypothesis that expression dynamics of sperm nuclear proteins, post-translational modifications (PTM) of sperm Histone 4 (H4), and seminal plasma metabolome are associated with bull fertility and sperm freezability (P = 0.043). Flow cytometry experiments were conducted to quantify H4 and acetylated histone 4 (H4ac) in sperm from high and low fertility Holstein bulls. The analysis of flow cytometry experiments clarified that retained levels of H4ac in bull sperm are associated with bull fertility. In addition, gas chromatography-mass spectrometry (GC-MS) was applied to ascertain the amino acid concentration of seminal plasma from bull semen with various freezability. A total of 21 amino acids and isomers were identified, and phenylalanine was positively associated with sperm post-thaw viability (r = 0.57, P-value = 0.043). Lastly, a quantitative western blotting experiment was utilized to ascertain relative quantification of sperm nuclear proteins including protamine 1 (PRM1), protamine 2 (PRM2), Histone 3 (H3), and H4. Also, sperm functional parameters including acrosome reaction, DNA fragmentation index, PAWP expression were analyzed using flow cytometry. In addition, immunocytochemistry experiments were applied to analyze sperm chromatin decondensation ability. The analyses of western blotting experiments revealed that the relative abundance of PRM2 in poor freezability sperm (PF) was greater than those in good freezability sperm (GF) (P = 0.0259). The relative abundance of retained H3 was greater in PF bulls than in GF bulls (1.02 ± 0.005 and 0.969 ± 0.021, respectively; P = 0.0272). There was a positive correlation between the abundance of retained H4 and sperm decondensation state (r = 0.71, P = 0.05). These results are important because they can help advance fundamental andrology and the assisted reproductive technologies both for cattle and other mammals, including humans and endangered species.