To investigate the potential protective effect of heat shock protein 70 (Hsp70) during heat stress (HS) in chicken primary myocardial cells (CPMC), a cellular model of low expression of Hsp70 was established using 200 µM quercetin, a specific inhibitor of Hsp70. Comparative analyses were done among a HS group, Hsp70 low expression (HS+Quercetin) group and quercetin treated only group (Quercetin) during different durations of HS (0, 1, 2, 3 and 5 h). Inhibition of Hsp70 expression in quercetin treatment groups was detected, and suggested that Hsp70 expression was inhibited significantly. Levels of enzymes associated with cardiac damage were measured. In the Hsp70 low expression group, levels of these enzymes were elevated significantly compared with HS group, quercetin alone didn't elevate the level of these enzymes, The Hsp70 low expression group had twofold greater apoptosis compared with the HS group after 5 h of HS which was consistent with the results of Cleaved caspase‑3 protein, no obvious apoptosis was detected in quercetin group. Levels of caspase-3 and -9 activities were significantly higher in the Hsp70 low expression group, no differences of apoptosis inducing factor (AIF) in cell nucleus were observed between two groups suggested that inhibition of Hsp70 in CPMC increased the percentage of apoptosis may involve a mitochondrial pathway but AIF was not included. Expression of Bax with Bcl‑2 and their downstream cytochrome c in two groups confirmed our hypothesis. Our findings suggest that in CPMC, Hsp70 may have a cytoprotective role during HS that may act via a mitochondrial pathway.