This study aimed to investigate the relationship between the sperm quality and the osteopontin (OPN) concentration in the prostates of Malakli shepherd dogs. Ejaculates were collected once by digital manipulation from 39 male dogs aged between 2 and 4 years and older. The first and third fractions of the ejaculate were centrifuged at 5000× g for 30 min, and supernatants were stored at −80 °C for further analysis of OPN using a double-antibody sandwich method (SEA899CA, Cloude-Clone Corp, Houston, TX, USA). Meanwhile, the second fractions were evaluated for sperm motility, concentration, viability, and rate of abnormal spermatozoa (head, acrosome, midpiece and tail abnormalities). The average concentration of OPN was 8.7 ± 5.2 ng/mL, and it differed significantly between the 1st 10.4 ± 5.3 ng/mL and 3rd 7.4 ± 5 ng/mL fractions. According to ROC (receiver operating characteristic curve) analysis, the OPN concentration had a better diagnostic ability for sperm motility (p < 0.001) than for the rate of abnormal spermatozoa (p < 0.05). Additionally, the OPN concentration was negatively correlated with poor sperm morphology and motility. In conclusion, the OPN concentration in prostate-derived secretions may be a possible marker of sperm quality in dogs. Further research could explore the involvement of OPN in sperm motility during cryopreservation and in vivo fertility.