Tuberculosis (TB) is an infectious disease displaying a multifactorial pathology. The immunomodulatory role attributed to steroid hormones, such as vitamin D₃ (VD₃) and 17β-estradiol (E₂), highlighted the importance of these hormones against Mycobacterium tuberculosis (Mtb) infection. In order to understand their influence upon gene expression of immune and inflammatory responsive genes against Mtb we tested it in vitro using peripheral blood mononuclear cells (PBMCs). Cells were pretreated with VD₃ (50 ng/mL) or E₂ (100 nM/mL) and co-cultured with H37Rv Mtb or stimulated with lipopolysaccharide from Escherichia coli (LPS). After 24 h and 72 h of co-culture the Mtb viability in macrophages test was performed, as well the total RNA isolation for gene expression analysis by RT-qPCR of the following target genes: NLRP3, DC-SIGN, IL-1β, and IL-10. We also measured IL-10, TNF, IFN-γ, IL-4, IL-6, and IL-2 supernatant levels. As the main results, we found that VD₃ and E₂ downregulated the expression of inflammatory genes NLRP3, IL-1β, and IL-10 expression in Mtb co-cultured cells. Finally, VD₃ treatment increased the release of the cytokine IFN-γ in Mtb-infected cells, while E₂ treatment inhibited the release of IL-10, TNF, IFN-γ, and IL-6. Therefore, we report an immunogenetic influence of VD₃ and E₂ upon Mtb co-culture.