Citrulline has anti-inflammatory properties and exerts beneficial effects on various impaired functions in aging. However, there are few data on citrulline action on immune function in aged populations. The objective of the study was to evaluate citrulline ability, after in vivo and in vitro administration, to modulate macrophage functions in aged rats and the possible pathways involved. Twenty-one-month-old Sprague-Dawley rats (n = 27) received a citrulline supplementation at 5 g/kg/d for 5 days, or an isonitrogenous diet, and peritoneal macrophages were cultured with or without LPS. In the in vitro study, macrophages from 22-month-old rats (n = 16) were cultured with or without LPS, citrulline and inhibitors of different inflammatory pathways (n = 8/conditions). Nitric oxide (NO) and tumor necrosis factor α (TNFα) production were measured in both in vivo and in vitro studies. Citrulline decreased NO production variability by peritoneal macrophages after in vivo administration (p = 0.0034) and downregulated NO production by 22% after in vitro administration (95% CI: [6%; 35%]; p = 0.0394), without any direct effect on TNFα production. None of the transductional pathways explored seem to be involved. Citrulline slightly modulates NO production in vivo and in vitro, suggesting a possible action through modulation of arginine metabolism in macrophages rather than a direct transductional effect. The pleiotropic effects of citrulline in aging could be due, at least in part, to the anti-inflammatory effect of citrulline.