The influences of the addition of glycolipid analogues to the vesicle membrane composed of nonionic surfactant Span80 on the membrane characteristics of the vesicle were investigated. The analogues examined were as follows: N-[O-(D-glucopyranosyl)-6-oxyhexyl]-3,5-bis(dodecyloxy)benzamide (Glc-L6-2), N-[O-(D-glucopyranosyl)-6-oxyhexyl]- 3,4,5-tris(dodecyloxy)benzamide (Glc-L6-3) and 3,4,5-tris(dodecyloxy)benzyl-D-glucopyranoside (Glc-Bn-3). The membrane fluidity of Span80 vesicle was compared with some phospholipid liposomes by measuring the fluorescence anisotropy (1/P value) with 1,6-diphenyl-1,3,5-hexatrien. The fluidity of Span80 vesicle was higher than those of the liposomes because of very lower phase-transition temperature (–40 ℃) of the Span80 vesicle. The addition of each glycolipid analogue decreased the membrane fluidities of the Span80 vesicles with the order of the additive systems of Glc-L6-2, Glc-L6-3 and Glc-Bn-3. The effects of the additive glycolipid analogues to Span80 vesicle on the formation ratio and the initial burst of the vesicle were evaluated by measuring the leakage of the Brilliant blue tracer entrapped in the vesicle. The addition of either Glc-L6-3 or Glc-Bn-3 of 0.2 ～ 0.3 wt% also enhanced the vesicle formation and inhibited the initial burst of the vesicle. Thus, the fluidity, formation and initial burst of Span80 vesicle were controlled by incorporating the glycolipid analogues to the vesicle membrane.