Colibacillosis is an infectious disease produced by avian pathogenic E.coli associated with reduced productivity, high mortality and condemnation rates as well as increased treatment costs. Colistin has become the predominant treatment option against multidrug resistant (MDR) Gram-negative bacteria. Nevertheless, progression of renal damage caused by injection of colistin frequently hinders the attainment of ideal therapeutic dosages. The present work aimed to formulate colistin-loaded niosomes (CLN) to improve efficacy and decrease toxicity of colistin as a potential treatment against avian pathogenic E.coli in broiler chicks. The CLN in tween 60, cholesterol and dihexadecyl phosphate in the molar ratio of 1:2:0.1 was chosen as an efficient carrier for delivering colistin. The minimum inhibitory concentration for CLN was approximately 12 times lower than that of free colistin with enhanced pharmacokinetic parameter, demonstrating the higher efficacy of CLN. Efficacy and safety of CLN were investigated in vivo using an experimental bird model using E. coli. In contrast to control positive group, serum albumin, total protein and creatinine concentrations were significantly lower following parenteral CLN administration. Histopathological examination of the kidney demonstrated that CLN inhibits nephrotoxic effects when compared to free colistin. Additionally, microscopical examination of liver, lung, and heart samples demonstrated the safety of the CLN. In contrast to colistin sulphate, niosomal colistin demonstrated superior pharmacological activity and efficacy suggesting that administering parenteral CLN is more effective and safer than conventional colistin.