Abstract The aim of this study was to investigate potentialities of poly(dl-lactide–co-glycolide) (PLGA) microspheres for the delivery of small interfering RNAs (siRNAs) against tumor necrosis factor α (TNF-α) to achieve prolonged and efficient inhibition of TNF-α for the treatment of rheumatoid arthritis (RA). PLGA microspheres were prepared by a modified multiple emulsion–solvent evaporation method. The formulations were characterized in terms of morphology, mean diameter and siRNAs distribution, encapsulation efficiency, and in vitro release kinetics. The efficiency of this system was then evaluated both in vitro and in vivo using the murine monocytic cell line J774 and a pre-clinical model of RA, respectively. siRNA-encapsulating PLGA microspheres were characterized by a high encapsulation efficiency and a slow and prolonged anti-TNF-α siRNAs. Our results provide evidence that, upon intra-articular administration, PLGA microspheres slowly releasing siRNAs effectively inhibited the expression of TNF-α in arthritic joints. Our system might represent an alternative strategy for the design of novel anti-rheumatic therapies based on the use of RNA interference in RA.