Effects of monoglyceride (MG) on the properties of WPI-stabilized emulsions and the bioavailability of encapsulated β-carotene were investigated. MG-structured emulsions showed reduced surface charge, higher viscosity, and better creaming stability than an emulsion without MG. Exposure of emulsions to GIT digestion led to significant changes in droplet size and interfacial properties. In vitro bioavailability of β-carotene in 1% MG (63.9%) and 2% MG (77.1%) structured emulsions were higher than that in emulsion without MG (53.4%) (p < 0.05). All MG emulsions demonstrated a better cellular uptake of β-carotene by Caco-2 cells than the emulsion without MG (p < 0.05). A significant increase in the cellular uptake of β-carotene with increasing MG content was observed, increasing from 0.109 μg/well for the 0.5% MG emulsion and up to 0.138 μg/well for 2% MG emulsion. The findings in this study confirm the potential of the MG-structured emulsions as novel carriers for lipophilic nutrients with improved stability and bioavailability.