Rationale <p>Wheel running is a highly motivated behavior in rodents and is often used as a natural reinforcer. The adenosine A_2A receptor is abundantly expressed in the nucleus accumbens (NAc) and plays a key role in the regulation of motivation and reward-related behaviors. However, its involvement in motivation for wheel running remains unclear.</p> Objectives <p>This study aimed to investigate how systemic and intra-NAc pharmacological manipulation of A_2A receptors affects motivation for wheel running in mice.</p> Methods <p>We examined the effects of systemic administration of the A_2A receptor antagonist istradefylline and the A_2A receptor agonist CGS21680 on voluntary wheel-running activity and general locomotor activity. We also assessed the effects of these drugs on operant conditioning tasks in which wheel-running served as a reward. Furthermore, we infused CGS21680 directly into the NAc to examine site-specific effects.</p> Results <p>Systemic istradefylline administration increased voluntary wheel-running activity and general locomotion. In contrast, CGS21680 decreased voluntary wheel-running without affecting locomotion. In the operant task, systemic CGS21680 significantly reduced the operant response, a measure of appetitive behavior, without altering running duration, which reflects consummatory behavior. Similarly, intra-NAc infusion of CGS21680 suppressed appetitive behavior.</p> Conclusions <p>These findings demonstrate that, in male mice, A_2A receptor activation selectively suppressed the appetitive component of wheel-running motivation, whereas A_2A receptor blockade did not. Together, our results highlight the crucial role of NAc A_2A receptors in regulating motivation for wheel running and suggest the potential therapeutic application of A_2A receptor agonists for treating maladaptive behavioral over-engagement.</p>