The influence of interfacial composition on the in vivo digestibility of emulsified and encapsulated lipids was investigated. An electrostatic layer-by-layer deposition technique was used to prepare soybean oil-in-water emulsions that contained lipid droplets coated by lecithin or by lecithin–chitosan. Thirty six 4-week-old male mice were divided into four groups and fed treatment diets for 4 weeks; atherogenic diets supplemented with (A) non-emulsified fat, without chitosan (control), (B) non-emulsified fat, with chitosan, (C) emulsified fat, without chitosan, or (D) emulsified fat encapsulated by chitosan. There were no differences in body weights, food intake, major organ weights, or fecal fat contents between all treatment groups, where total fat absorption was >90%. The results suggest that encapsulation of lipids by chitosan does not inhibit their in vivo digestibility, even though previous studies indicate that chitosan does inhibit their in vitro digestibility. Consequently, it should be possible to use chitosan to microencapsulate lipids and lipid-soluble components without compromising their bioavailability, although further human studies are needed to confirm this.