The present work describes preparation and in vitro, characterization of niosomes containing 5-fluorouracil (5-FU) using non-ionic surfactant series of sorbitan esters (Spans) by thin film hydration method. Niosomes prepared from Span 40 and Span 60 exhibit higher entrapment of 5-FU as compared Eo niosomes prepared from Span 80 and Span 85. Cholesterol increases the entrapment of 5-FU into niosomes. Dicetyl phosphate reduces the size of niosomes. The leakage of 5-FU from niosomes reduces on freeze drying.