Mammalian spermatozoa are known to respond to a temperature gradient by thermotaxis, with an end result of accumulation in the warmer temperature (1). The behavioral mechanism that underlies thermotaxis is not known. Our aim in this study was to reveal this mechanism in human spermatozoa. We exposed spermatozoa on a microscope slide to a rapid change of temperature from 31 to 37°C and vice versa, achieved by a thermoregulated microscope stage, and recorded the swimming of the spermatozoa under the microscope at 75 and 2000 frames/s. The spermatozoa responded to the temperature rise by increased velocity (~25% on average), higher linearity, lower side-to-side amplitude of head displacement, and a reduced degree of hyperactivation. These changes were a response to both the absolute temperature and the temperature gradient, as revealed from partial adaptation upon reaching 37°C and from control experiments at constant temperatures. Essentially the opposite trend was observed when the spermatozoa were exposed to a decreasing temperature gradient. These results suggest that, as in sperm chemotaxis, human spermatozoa sense a temperature gradient temporally rather than spatially. They further suggest that spermatozoa respond to the temperature gradient by modulating the frequency of directional changes and hyperactivation events, being higher in a decreasing gradient and lower in an increasing gradient. Similar characteristics of behavior were observed when spermatozoa were exposed to a temperature gradient in a more viscous medium, at viscosity similar to that believed to exist in vivo. The directional changes and hyperactivation events stimulated by the temperature gradient are similar to those reported for the behavior of human spermatozoa in a chemoattractant gradient (2), suggesting a common mechanism of behavior for both chemotaxis and thermotaxis.