The role of heat shock protein in reproduction is widely known as a molecular chaperone in aiding and repairing protein formation when stress occurred. The present objectives were to evaluate the effect of different thawing temperature and time on the expression of HSP70 gene expression and the capacitation status in cryopreserved bovine spermatozoa. Briefly, fresh ejaculates were obtained from three different adult bulls. The semen then underwent a sperm washing technique known as Magnetic Activated Cell Sorting System (MACS) and later on, cryopreserved. The sperm- containing straws were then thawed at five different thawing temperatures and time post-cryostorage; 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s and 80°C for 5 s. The RNA was extracted from each of the sperm’s pellets and converted to cDNA prior to the qPCR process. Capacitation status was then determined by means of CTC assay. The results showed that after the process of amplification, there is a significant different of HSP70 gene expression in MACS process samples when the thawing process was performed at 37°C for 30 s, with p<0.05. Furthermore, the CTC assay also showed that thawing at the same temperature gave less capacitated spermatozoa with p<0.05. As a conclusion, MACS yield spermatozoa with a better expression of HSP70 gene and less capacitated spermatozoa when thawing was done at 37°C for 30 s.