Novel pH-sensitive nanospheres designed for colon-specific delivery were prepared using polymeric mixtures of poly (lactic-co-glycolic) acid (PLGA) and methacrylate copolymers. Budesonide (BSD), a topically active corticosteroid, was entrapped as a model drug. The therapeutic efficacy of the prepared nanospheres was assessed using the trinitrobenzenesulfonic acid (TNBS) colitis rat model, in comparison with conventional enteric microparticles. In addition, the colon targeting properties, systemic bioavailability, and specific uptake by the inflamed colon mucosa were evaluated using coumarin-6 (C-6) loaded nanospheres. The prepared nanospheres showed strongly pH-dependent drug release properties in acidic and neutral pH media followed by a sustained release phase at pH 7.4. Animal experiments revealed the superior therapeutic efficiency of BSD nanospheres in alleviating the conditions of TNBS-induced colitis model. The in-vivo studies using C-6-loaded nanospheres displayed higher colon levels and lower systemic availability of the fluorescent marker when compared with simple enteric coating. Moreover, quantitative analysis of the fluorescent marker and confocal laser scanning studies showed strong and specific adhesion of the nanospheres to the ulcerated and inflamed mucosal tissue of the rat colon. In conclusion, the proposed nanosphere system combined the properties of pH-sensitivity, controlled release, and particulate targeting that could be useful for colon-specific delivery in inflammatory bowel disease.