Oral delivery of insulin is challenging and must overcome the barriers of gastric and enzymatic degradation as well as low permeation across the intestinal epithemlium. The present study aims to develop a liposomal delivery system that contains glycocholate as an enzyme inhibitor and permeation enhancer for oral insulin delivery. The liposomes containing sodium glycocholate (SGC-liposomes) were prepared by a reversed-phase evaporation method followed by homogenization. The particle size and entrapment efficiency of rhINS-loaded SGC-liposomes can be easily adjusted by tuning the homogenization parameters, phospholipid/SGC ratio, insulin/phospholipid ratio, the water/ether volume ratio, interior water phase pH and the hydration buffer pH. The optimal formulation showed an insulin entrapment efficiency of 30  2% and a particle size of 154  18 nm. The conformational study by circular dichroism spectroscopy and bioactivity study confirmed the preserved integrity of rhINS against the preparative stress. Transmission electron micrographs revealed a near spherical and deformed structure with discernable lamellar for SGC-liposomes. SGC-liposomes showed better protection of insulin against enzymatic degradation by pepsin, trypsin and α-chymotrypsin than liposomes containing bile salt counterparts of sodium taurocholate and sodium deoxycholate. In conclusion, SGC-liposomes showed promising in vitro characteristics and have potential to be used to delivery insulin orally.