Vitamin D deficiency is a cause of concern across the world. It is a fat-soluble vitamin and exhibits two molecular forms (D2 and D3). The aim of the present study is the development and optimization of vitamin D3 loaded PLGA microspheres by using Box Behnken Design. Additionally, to enhance nasal permeation these microspheres were dispersed in the thermoreversible in-situ gel. Microspheres revealed particle size in the range of 9182 ± 1 nm with 75.5 ± 0.5%EE of vitamin D3. Polydispersity index and zeta potential of microspheres were found 0.509 and −7.56 mV, respectively confirming uniformity and colloidal stability. FTIR and DSC studies revealed no interaction between excipients and vitamin D3. Scanning electron microscopy demonstrated spherical morphology of microspheres. The thermoreversible in-situ dispersed with vitamin D3 loaded microspheres exhibit the gelation temperature between 35 °C to 37 °C. The viscosity of the gel was 35698–5032 cps. The gel strength was found 1.1 ± 0.2 mJ with hardness (27 g), adhesiveness (22 g), and stringiness (3.2 mm) measured by a texture analyzer. In vitro dissolution study revealed that microspheres showed 79% VD3 release. Whereas microspheres dispersed gel showed 69% of VD3 release up to one week as compared to an oily solution. The mucoadhesion of gel was found 19600 dyne/cm2 suggesting the extraordinary high mucoadhesive property. Ex vivo permeation study through goat’s nasal mucosa suggests that gel formulation showed 2.5 folds high permeation than microspheres and solution. Thus, it could be a potential drug delivery system for the delivery of vitamin D3 via nasal route.