Vitamin D (VD) plays a crucial role in age-related diseases, and its influence on cellular senescence (CS) could help clarify its function in aging. Considering VD’s pleiotropic effects and the heterogeneity of CS, we utilized single-cell RNA sequencing (scRNA-seq) to explore these dynamics across multiple tissues. We analyzed three murine tissue datasets (bone, prostate, and skin) obtained from public repositories, enriching for senescent gene signatures. We then inferred gene regulatory networks (GRNs) at the tissue and cell-type levels and performed two cell communication analyses: one for senescent cells and another for interactions between senescent and non-senescent cells. VD supplementation significantly decreased senescence scores in the skin (p = 3.96e-134) and prostate (p = 1.56e-34). GRN analysis in the prostate revealed an altered macrophage-fibroblast regulatory relationship. In bone, distinct aging-related modules emerged for different bone lineages. In skin, contrary differentiation patterns between suprabasal and basal cells were observed. The main VD-modulated pathways were involved in inflammation, extracellular matrix remodeling, protein metabolism, and translation. VD reduced fibroblast-macrophage interactions in the prostate and skin but increased overall cellular crosstalk in bone. Our findings demonstrate that VD alleviates CS burden across tissues by modulating inflammation and metabolic processes and promoting differentiation. Key aging-related genes modulated by VD were linked to anabolism and cellular differentiation, suggesting VD’s potential for therapeutic interventions targeting age-related diseases.