The aim of this study was to examine the effect of estradiol (E2) on the thermoregulatory responses induced by cinnamaldehyde, a component extracted from cinnamon at 16 °C or 27 °C. The thermoneutral and subneutral experiments were performed to evaluate the augmented effect of cinnamaldehyde by cold exposure and the effect of cinnamaldehyde itself. Ovariectomized rats were implanted with a silastic tube with or without E2 (E2(+) and E2(-) groups), and data loggers into the peritoneal cavity. After the application of 30% cinnamaldehyde or vehicle into the skin of the whole trunk of rats, the rats were exposed to 16 °C or 27 °C for 2 h. Body temperature (Tb) and tail temperature (Ttail) were measured using a data logger and thermography. After exposure, blood samples were obtained, and plasma catecholamine concentration were measured by high-performance liquid chromatography. In the E2(-) group exposed to 27 °C, the change in Tb in rats applied cinnamaldehyde was significantly lower than that of rats applied vehicle. The change in Ttail in rats applied cinnamaldehyde was significantly lower than that of rats applied vehicle in both E2(-) and E2(+) groups at 27°C and 16°C. Plasma catecholamine concentration at 27°C was not different among the groups. E2 might not affect thermoregulatory responses induced by cinnamaldehyde application; however, it decreased Ttail in female rats.