The aim of the present study was to identify the correlation between expression of heat shock protein 47 (Hsp47) and stress injury in heat-stressed myocardial cells and to compare variations in Hsp47 expression in rat myocardial cells exposed to different heat stress for varying periods in vitro and in vivo. Exposure to heat stress at 42°C resulted in similar induction patterns of the heart damage-related enzyme aspartate aminotransferase in the supernatants of H9c2 cells and in the serum of rats. Histological analysis revealed that both H9c2 cells and heart tissues displayed cellular degeneration in response to different periods of heat stress. Hsp47 was constitutively expressed in the cytoplasm of H9c2 cells at all time points during heat stress, which was consistent with observations in heart fibers in vivo. Immunoblotting analysis revealed no significant difference between the expression of Hsp47 in H9c2 cells and heart tissue. However, the expression of hsp47 mRNA in response to heat stress was significantly increased in H9c2 cells at 60 min (P < 0.01) and 100 min (P < 0.01), which was comparable to that at 100 min (P < 0.01) in the rat heart. Thus, Hsp47 was elevated significantly after hyperthermia at the mRNA level but not at the protein level both in vitro and in vivo. The results suggest that Hsp47 turnover may increase during heat stress or that Hsp47 consumption exceeds its production.