1,25 Dihydroxy vitamin D3 (vitamin D3) is an immunomodulator and its deficiency has been associated with susceptibility to tuberculosis. We have studied the immunoregulatory role of vitamin D3 on various chemokine expression in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 21 pulmonary tuberculosis (PTB) patients and 24 healthy controls (HCs) were cultured for 48h with culture filtrate antigen (CFA) of Mycobacterium tuberculosis with or without vitamin D3 at a concentration 1×10−7M. The relative mRNA expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), macrophage inflammatory protein-1α (MIP-1α, CCL3), macrophage inflammatory protein-1β (MIP-1β, CCL4), and regulated upon-activation, normal T cell-expressed and secreted (RANTES, CCL5) and IFN-γ inducible protein-10 (IP-10, CXCL10) chemokines were estimated from 48h old macrophages using real-time polymerase chain reaction (RT-PCR). The culture supernatants were used to estimate the various chemokines including monokine induced by IFN-γ (MIG, CXCL9) levels using cytometric bead array. In HCs, vitamin D3 significantly suppressed the MCP-1 mRNA expression of CFA stimulated cells (p=0.0027), while no such effect was observed in PTB patients. Vitamin D3 showed no significant effect on MIP-1α, MIP-1β and RANTES in both the study groups. The CFA induced IP-10 mRNA and protein expression was significantly suppressed by vitamin D3 in both the study groups (p<0.05). A similar suppressive effect of vitamin D3 was observed with MIG protein in healthy controls (p=0.0029) and a trend towards a suppression was observed in PTB patients. The suppressive effect of vitamin D3 is more prominent in CXC chemokines rather than CC chemokines. This suggests that vitamin D3 may down regulate the recruitment and activation of T-cells through CXC chemokines at the site of infection and may act as a potential anti-inflammatory agent.