Canadian First Nations (FN) population experiences a high burden of tuberculosis. Vitamin D is known to enhance the expression of innate immune effectors, including cathelicidin LL-37, for protection against infections. In this study we performed longitudinal analyses to investigate the impact of vitamin D supplementation on macrophage responses to Mycobacterium tuberculosis (Mtb) lipoprotein (TLR2/1L), in Canadian Dené FN participants compared to Caucasian participants. Serum 25(OH)D and LL-37 levels were evaluated by ELISA. Transcriptional responses and protein expression of TLR2/1L-induced LL-37 and other innate immune cytokines were monitored in monocyte-derived macrophages (MDMs) before and after 8 months of vitamin D supplementation. In this study we showed that serum levels of LL-37 decreased after vitamin D supplementation in both Dené and Caucasian participants. There was no difference in TLR2/1L-induced LL-37 expression in MDMs in the two groups, either pre- or post-vitamin D supplementation. However, vitamin D supplementation markedly enhanced TLR2/1L-induced responses in MDMs e.g. IL-6, IL-12 and IL-23 among Caucasians but not in the Dené participants. In contrast, after vitamin D supplementation TLR2/1L-induced responses e.g. IL-1β, IL-8 and IL-12 were significantly reduced in the Dené MDMs. These results indicate that vitamin D supplementation enhanced TLR2/1L-induced innate immune macrophage responses in the Caucasian but not in the Dené participants. We hypothesize that cytokines may be differentially regulated in Canadian FN compared to Caucasians, in particular those that influence Th-1 and Th-17 responses required for the control of Mtb.