Habituation is ubiquitous throughout the animal kingdom. Nonetheless, there are no studies that report habituation of the rattling behavior in rattlesnakes. Ten Western Diamondback Rattlesnakes (Crotalus atrox) were stimulated to rattle in an automated apparatus at five-minute intervals until they failed to rattle for ten consecutive trials over four consecutive days. Group data are reported for the occurrence of rattling, latency to rattle, and duration of rattling. Both long-term and short-term habituation were documented, though there was notable variation within and between individuals. Differences within and among individuals over the four testing days were found. We recommend that future studies focus on proximate factors (e.g., sex, body temperature) that may influence the course of habituation of the rattle response.