Despite Latin America's rich biodiversity, active genetic material conservation programs are scarce. This study investigates potential freezability markers in both sperm and seminal plasma (SP) in Chino Santandereano, a Colombian Creole breed. Thirty ejaculates from ten Chino Santandereano bulls were cryopreserved and subsequently classified as of good (GFE) or poor (PFE) freezability according to their post-thaw total sperm motility (TMOT) and plasma membrane integrity (PMI). Sperm cholesterol and NPC2 protein levels in SP were measured prior to cryopreservation using colorimetric detection, densitometric quantification, and Western blot. Sperm functional competence (SFC), a functional test, was evaluated before and after cryopreservation. Correlation and t-student tests, a binomial regression model and two multiple linear regression models were utilized to evaluate the predictive power of NPC2 protein and sperm related variables. Two bands (14 kDa and 15 kDa) for NPC2 protein were identificated. NPC2 14 kDa is associated with a significant decrease in freezability (P <
0.01). The TMOT in fresh semen highly significant (P <
0.01) for sperm freezability and the interaction between NPC2 14 kDa and cholesterol content in sperm was significant (P <
0.01). Correlation between SFC and TMOT and PMI post-thaw (P <
0.01), along with the significantly different values between PFE and GFE (P <
0.05) were observed. We can conclude that NPC2 protein as a marker for sperm freezability of Chino Santandereano bull, and this depends on sperm content of cholesterol. In addition, our findings support the utility SFC combined tests in providing a precise assessment of sperm freezability.