The present study was conducted to investigate whether dietary sanguinarine (Sangrovit^®, SGV) could affect growth performance, relative organ weigths, gut microbiota, serum cholesterol levels, and malondialdehyde contents of leg meat in broiler chickens. A total of 840 day-old male broiler chicks (Ross breed) was randomly placed on 28 floor pens with rice straw as a bedding and subjected to one of four experimental diets; corn-soybean meal based control diet, the control diet added with avilamycin at 10 ppm as growth promoter (AGP) and SGV at the level of 20 (SGV20) or 50 ppm (SGV50). The final body weight (BW), daily BW gain, and feed conversion ratio (FCR) were significantly improved (P<0.05) in broiler chickens fed either AGP, SGV20 or SGV50 compared with the control diet-fed chickens. Compared with the control group, relative jejunal weight was significantly lowered (P<0.05) in SGV20-fed chickens and relative jejunal or ileal length was significantly increased (P<0.05) in all SGV-fed chickens. Dietary SGV20, but not AGP, altered gut microbiota (especially increase in cecal lactic acid bacteria) compared with the control diet-fed chickens. Total cholesterol of broiler chickens fed on a diet containing SGV20 or SGV50 vs. the control diet was significantly reduced. Finally, the content of malondialdehyde in thigh meats as an indicator of lipid peroxidation was significantly lowered (P<0.05) by dietary SGV compared with that seen in the control chickens. In conclusion, our study clearly reveals that supplementation of SGV into the broilers’ diet at 20 or 50 ppm improved growth performance and altered various biological and physiological parameters such as relative organ weights, serum cholesterol levels, gut microbiota, and meat qualities in broiler chickens.