This study aimed to improve the anticancer activity of letrozole through a niosomal formulation. Optimized niosomal formulation of letrozole was achieved by response surface methodology (RSM). The niosomes were well-characterized by several methods. The anticancer activity and its mechanism were studied in MCF-7 and MDA-MB-231 breast cancer cells. The release of the drug from the niosomes was according to the Kors Meyer-Peppa kinetic model. The niosomes were stable with high encapsulation efficiency. Significant higher anticancer activity and more induction of apoptosis were obtained for niosomal letrozole. Results indicated that niosomes could be a promising drug carrier for delivery of letrozole to breast cancer cells.