The endocannabinoid system (ECS) is involved in regulation of various physiological functions, including locomotion, antinociception, emotional states, and motivated behaviors. The ECS has been implicated in regulation of voluntary wheel running in mice via actions at the cannabinoid receptor-1 (CB1). Previously, we showed that four replicate lines of mice bred for high levels of voluntary wheel running (high-runner or HR lines) sex-specifically (females only) decreased running in response to antagonism of the CB1 receptor, as compared with four unselected Control lines. Here, we administered a CB1 receptor agonist, WIN 55,212-2 (WIN). We predicted that if CB1 activation is involved in the regulation of voluntary wheel running, then HR mice would show a greater response to CB1 agonism. Following our previous protocols, mice from generation 53 were acclimated to running wheels for 24 days, then received, in random order, either an intra-peritoneal injection of vehicle or a low (0.5 mg/kg), medium (1 mg/kg) or high dosage (3 mg/kg) of WIN. Each mouse received an injection and then experienced two nights without injections, for a total period of 12 days. Response to WIN was quantified as wheel revolutions, time spent running, and average running speed in the 10-120 min immediately following injection. Injection decreased wheel revolutions in all mice, but male HR mice decreased their running to a greater degree relative to Controls in response to the high dose of WIN over the entire period analyzed, whereas HR females showed a differential response relative to Controls only in the latter 70-120 min post-injection. These results, in conjunction with our previous study, show that (a) aspects of endocannabinoid signaling have diverged in four lines of mice bred for high levels of voluntary exercise and (b) male and female HR mice differ from one another in CB1 signaling as it relates to wheel running.