Rutin is a bioflavonoid with strong antioxidant activity. To investigate the regulatory roles of rutin in various functions in crustaceans, we examined physiological (haemolymph glucose, lactate, and lipid) and innate non-specific immune responses (total haemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (release of superoxide anion, O2−) and superoxide dismutase (SOD) activity) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with rutin extracted from Toona sinensis at 10, 20, or 50 μg g−1. Results showed that PO activity and respiratory burst of L. vannamei increased obviously (P < 0.05) when injected with rutin at a dose of 20 and 50 μg g−1 after 12 and 24 h, respectively. Both the THC and SOD activities of experimental and control groups revealed no significant difference at all doses. L. vannamei injected with rutin at either dose maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge after 12–36, 24–48, and 24–60 h, respectively. The survival rate of L. vannamei that received rutin at either dose was significantly higher than that received saline after 48–72 h. It was, therefore, concluded that the immune ability and resistance against V. alginolyticus infection of L. vannamei receiving rutin at ≥10 μg g−1 increased.