There is a growing interest in co-encapsulating multiple species to harness potential synergy between them, enhance their stability and efficacy in various products. The aim of this work was to co-encapsulate vitamin D3 and rutin inside chitosan-zein microparticles using a simple and easily scalable process for food fortification. This was achieved via anti-solvent precipitation coupled with spray-drying. Free-flowing powders of spherical microparticles with wrinkled surface and particle size < 10 μm were obtained. The encapsulation efficiency was 75% for vitamin D3 and 44% for rutin and this could be attributed to their different molecular size and affinity to the aqueous phase. The physicochemical properties were characterized by X-Ray powder diffraction and Fourier transform infrared spectroscopy. The two crystalline bioactive compounds were present in the microparticles in amorphous form, which would allow for better bioavailability when compared to non-encapsulated crystalline solid. Therefore, the obtained microparticles would be suitable for use as food ingredient for vitamin D3 fortification, with the co-encapsulated rutin acting as stability and activity enhancer.