This study was carried out to determine the effect of L-arginine (L-Arg) levels in diet at the starter, grower and finisher phases on immune response, organ development, nitric oxide (NO) metabolism and colon motility in broilers. A total of 500 one-day-old Ross-308 broiler chickens of mixed sex were separated into one Arg-deficient group and four experimental groups. Each group was then divided into five subgroups of 20 birds each. Arginine deficient group for all phases was fed a basal diet which contained 10% less L-Arg than optimum Arg requirement recommended by the breeder. Experimental groups were fed a basal diet supplemented with L-Arg which was progressively 10% increased in groups. Thus, the diet contained 90, 100, 110, 120 and 130% of optimum Arg requirement for each phases in groups, respectively. The highest serum infectious bursal disease antibody titer (IBD) was observed in the experimental group which was fed the diet containing 110% L-Arg at grower phase (P < 0.05), whereas Newcastle disease antibody titer did not differ between groups. The relative weight of spleen increased in groups which were fed the diet containing 120 and 130% L-Arg at starter phase as compared to Arg-deficient group (P < 0.05). The group which was fed the diet containing 110% L-Arg showed highest relative weight of bursa Fabricii at grower (P < 0.05) and finisher (P < 0.01) phases. It was observed that serum nitric oxide (NO) concentration decreased in Arg-deficient group (P < 0.05). The amplitude of spontaneous colon contractility did not differ between groups at the end of all three phases. However, the frequency of spontaneous colon contractility in the Arg deficient group was higher at starter (P<0.05), grower (P < 0.01) and finisher (P < 0.05) phases. These results suggest that the supplementation of L-Arg at higher level than optimum Arg requirement in broiler diet has minimal effect on parameters investigated in the study. However, L-Arg-deficiency may negatively affect immune response and the motility of gastrointestinal system due to disruption of NO metabolism at three phases.