Self-microemulsifying drug delivery systems (SMEDDS) containing two types (Tween80® and Labrasol®) and two levels (low; 5% and high; 15%) of co-surfactants were formulated to evaluate the impact of surfactant phase on physical properties and oral absorption of oxyresveratrol (OXY). All formulations showed a very rapid release in the simulated gastric fluid (SGF) pH 1.2. After dilution with different media, the microemulsion droplet sizes of the Tween80®-based (˜26 to 36 nm) were smaller than that of the Labrasol®-based systems (˜34 to 45 nm). Both systems with high levels of surfactant increased the Caco-2 cells permeability of OXY compared to those with low levels of surfactant (1.4–1.7 folds) and the unformulated OXY (1.9–2.0 folds). It was of interest, that there was a reduction (4.4–5.3 folds) in the efflux transport of OXY from both systems compared to the unformulated OXY. The results were in good agreement with the in vivo absorption studies of such OXY-formulations in rats. Significantly greater values of Cmax and AUC0–10h (p < 0.05) were obtained from the high levels of Tween80®-based (Fr,0–10h 786.32%) compared to those from the Labrasol®-based system (Fr,0–10h 218.32%). These finding indicate the importance of formulation variables such as type and quantity of surfactant in the SMEDDS to enhance oral drug bioavailability.