The objective of this study was to compare the thermotolerances of ear fibroblasts derived from Holstein (H) and Taiwan yellow cattle (Y) and their apoptosis-related protein expressions with (1, 3, 6, 12, and 24 h) or without heat shock treatment. The results showed that the vaginal temperatures of Y (38.4–38.5 °C) were (P<0.05) lower than that of H (38.8 °C) during the hot season. The apoptotic rates of ear fibroblasts derived from Y (6 h: 1.1%; 12 h: 1.6%; 24 h: 2.6%) were lower (P<0.05) than those of cells derived from H (6 h: 1.8%; 12 h: 4.0%; 24 h: 6.9%), respectively, after heat shock (42 °C). The expression level of apoptosis inducing factor (AIF) in ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 6 h and 12 h, respectively. The level of cytochrome c of ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 1–12 h, respectively. The abundances of Caspase-3, Caspase-8 and Caspase-9 of ear fibroblasts derived from H were higher (P<0.05) than those of cells derived from Y after 12 h and 24 h of heat shock, respectively; the Bcl-2/Bax ratios of ear fibroblasts derived from H were lower (P<0.05) than those from Y-derived fibroblasts after heated for 1–24 h. The expression level of HSP-70 of Y-derived ear fibroblasts was also higher (P<0.05) than that from H after the same duration of heat shock treatments. Taken together, the thermotolerance of ear fibroblasts derived from Taiwan yellow cattle was better than that of cells derived from Holstein cattle.