Objective: This work aims to investigate the influence of various types and different contents of co-solvent on the stability and bioavailability of rapamycin formulated in self-microemulsifying drug delivery systems (SMEDDS). Methods: A series of SMEDDS of rapamycin were prepared with different co-solvents [including PEG 400/ethanol (F1), glycerol/ethanol (F2), propylene glycol (F3), glycerol formal (F4), transcutol P (F5)]. Drug stability in aqueous media at different pH values and in vitro dispersion of SMEDDS were investigated prior to bioavailability assessment. The storage stability of rapamycin in formulations was also evaluated. Results and discussion: The AUC values of rapamycin following oral administration of F1, F3-F5 to rats were significantly higher than those of Rapamune (R) and F0 (SMEDDS without co-solvent). Interestingly, a tendency toward increased bioavailability was seen in F1-F5, which presented the better drug stability in pH 1.2 aqueous media. However, a further increase of the content of co-solvent did not effectively improve the oral bioavailability of rapamycin. Compared with F0, F1-F5 presented significant improvement of drug storage stability. More specifically, the more-OH per unit mass co-solvent had, the better stability rapamycin presented in formulation. Conclusions: The data obtained in present study highlight the importance of co-solvents on the stability and bioavailability of rapamycin formulated in SMEDDS. Besides solubilizing drug and increasing the dispersion rate, co-solvent could markedly affect the stability of rapamycin whether in different aqueous media or during storage and contribute to the improved oral bioavailability; it can also appropriately decrease the content of surfactant without compromising the absorption of drug.