The Purpose of this work was to study the physicochemical characteristics of a series of melittin-loaded poly (d,l-lactide-co-glycolide) (PLGA) microspheres (MS) intended for intra-articular injection and the mechanism of melittin release. Melittin-loaded MS were prepared by a double emulsion solvent evaporation method and characterized in terms of drug loading, size, surface morphology, in vitro and in vivo in rat air-pouch (pseudo synovial fluid) drug release profiles. The effects of molecular weight of PLGA and osmotic pressure gradient between the inner and the outer water phase were investigated. The melittin MS were round in shape and with. the size in the range of 4-15 pm which is suitable for intraarticular injection. The encapsulation efficiency varied between 65 and 96%. In vitro melittin release occurred in typical tri-phase pattern. The in vitro release data were fitted to certain theoretical models and it was concluded that the erosion of polymer is the main mechanism that controls the release of melittin after initial burst release. Melittin could be released continuously and completely from the lower molecular weight PLGA for one month both in vitro and in vivo. There is a satisfactory correlation between in vitro and in vivo release behavior. The present work demonstrated that the lower molecular weight PLGA is the best candidate for a one month long-term sustained release dosage form of melittin.