Vitamin D has an immunomodulatory function and is involved in eliminating pathogens. Vitamin D deficiencies reported in Type 2 diabetes mellitus (T2DM) patients make them more susceptible to developing tuberculosis (TB). The macrophages are the immune cells that control intracellular pathogens by producing the antimicrobial peptide cathelicidin-LL37. This pathway involves TLR activation by pathogens, vitamin D receptor (VDR) ligation, and the enzyme 1α-hydroxylase Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1). However, it is not clear whether the biological actions of vitamin D are affected by high glucose concentrations. This study aimed to evaluate the vitamin D contribution in the expression of VDR and CYP27B1, involved in the conversion of an inactive to an active form of vitamin D in the infected macrophages usingas an infection model. The expression of LL37 and the nucleus translocation of VDR were evaluated as the readout of the response of vitamin D and determined if those processes are affected by glucose concentrations. Macrophages from healthy donors were cultured under glucose concentrations of 5.5, 15, or 30 mM, stimulated with vitamin D in inactive (25(OH)D) or active (1,25(OH)D) forms, and infected with. The vitamin D-dependent induction of LL37 and the expression of VDR and CYP27B1 genes were analyzed by qPCR, and VDR translocation was analyzed in nuclear protein extracts by ELISA.downregulated the expression of LL37 regardless of the glucose concentration, whereas VDR and CYP27B1 upregulated it regardless of the glucose concentration. After evaluating two concentrations of vitamin D, 1 nM or 1 μM, the high concentration (1 μM) was necessary to restore the induction of LL37 expression in-infected macrophages. High concentrations of the inactive form of vitamin D restore the infected macrophages' ability to express LL37 regardless of the glucose concentration. This finding supports the idea that vitamin D administration in patients with T2DM could benefit TB control and prevention.