Molecular-based biomarkers are believed to be more accurate in determining bulls' fertility and sperm's various fertility characteristics. The sperm acrosome-associated 1 (SPACA1) molecule, located in the anterior acrosomal and equatorial segments of the sperm head, is thought to be related to the function of binding and fusion between sperm and oocyte. This study aims to analyze the association of SPACA1 mRNA and protein with the fertility rate and semen quality of Bali bulls (Bos sondaicus) and assess its potential as a potential molecular biomarker determining bull fertility and sperm quality. Frozen semen from 20 Bali bulls was used in the research, which was then divided into two groups: high (HF) and low fertility (LF). SPACA1 mRNA abundance was analyzed using qRT-PCR, and SPACA1 protein abundance was analyzed using EIA. The semen quality parameters analyzed were motility (CASA), plasma membrane integrity (HOS test), sperm head morphology abnormalities (William staining), sperm viability (SYBR 14-PI), sperm capacitation (CTC assay), and sperm acrosome integrity (FITC-PNA). The results showed that there was a significant difference (P < 0.05) between high fertility (HF) and low fertility (LF) bulls, both in the abundance of SPACA1 at the mRNA, protein, and semen quality levels. Overall, the correlation test results showed a close relationship (P < 0.01) between the abundance of SPACA1 mRNA and protein with field fertility and various semen quality parameters tested in the study. A low level of SPACA1 molecules indicates low fertility and semen quality in Bali bulls. The SPACA1 molecule has the potential to be developed through further research to become a biomarker for determining fertility and semen quality in bulls.