Poly(lactic-co-glycolic acid) microspheres loaded with cholecalciferol (CL), more bioactive form of vitamin D was developed as an injectable controlled drug releasesystem and was evaluated for its feasibility of once a month delivery. The CL loaded microspheres (CL-MS) were prepared by simple oil in water (O/W) emulsion–solvent evaporation technique incorporated with a stabilizer, Tocopherol Succinate (TS). Different formulation as well as process parameters were investigated namely concentration of emulsifier, concentration of stabilizer and drug: polymer mass ratios. The prepared CL-MS were evaluated for particle size, drug loading, in–vitro drug release and in–vivo pharmacokinetics in rats. The optimized formulation was found to have a mean particle size of 28.62 ± 0.26 μm, Encapsulation Efficiency (EE) of 94.4 ± 5.4% and drug loading of 5.19 ± 0.29% with CL:TS ratio of 2:1. It was found that the EE drastically decreased (26 ± 5.9%) in the absence of stabilizer (TS) indicating its role in stabilization of CL during formulation. DSC and XRD studies indicated that CL existed in an amorphous structure in the polymer matrix. SEM of the CL-MS revealed the spherical morphology and confirmed the particle size. In–vitrorelease showed that the CL release from CL-MS followed near zero–order drug release kinetics over nearly 1 month. In–vivo pharmacokinetic study of CL-MS showed higher t1/2 (239 ± 27.5 h) compared to oily CL depot (32.7 ± 4.8 h) with sustained release of CL plasma concentration for 1 month. The labile CL could thus be effectively encapsulated and protected against degradation during microspheres formulation, storage and release in presence of stabilizer. This novel CL loaded PLGA MS is stable and may have great potential for clinical use.