Previous studies implicated the anti-inflammatory potential of the adenosine 2B receptor (A2BAR). A2BAR activation is achieved through adenosine, but this is limited by its very short t(1/2). To further define alternative adenosine signaling, we examined the role of netrin-1 during acute inflammatory peritonitis. In this article, we report that animals with endogenous repression of netrin-1 (Ntn1(+/-)) demonstrated increased cell count, increased peritoneal cytokine concentration, and pronounced histological changes compared with controls in a model of zymosan A peritonitis. Exogenous netrin-1 significantly decreased i.p. inflammatory changes. This effect was not present in animals with deletion of A2BAR (A2BAR(-/-)). A2BAR(-/-) animals demonstrated no change in cell count, i.p. cytokine concentration, or histology in response to netrin-1 injection. These data strengthen the role of netrin-1 as an immunomodulatory protein exerting its function in dependence of the A2BAR and further define alternative adenosine receptor signaling.