Prostaglandin (PG)E₂ is an arachidonic acid‐derived lipid mediator that plays an important role in inflammation and immunity. In this study, we demonstrate that PGE₂ suppresses basal and 1,25‐dihydroxy vitamin D₃ (VD₃)‐induced expression of hCAP18/LL‐37 via E prostanoid (EP)2 and EP4 receptors. In humans, VD₃ up‐regulates vitamin D receptor (VDR) expression and promotes transcription of the cathelicidin hCAP18/LL‐37 gene, whereas PGE₂ counteracts this effect. We find that PGE₂ induces the cAMP/PKA‐signaling pathway and enhances the expression of the inhibitory transcription factor cAMP‐responsive modulator/inducible cAMP early repressor, which prevents VDR expression and induction of hCAP18/LL‐37 in human macrophages. The negative regulation by PGE₂ was evident in M1‐ and M2‐polarized human macrophages, although PGE₂ displayed more profound inhibitory effects in M2 cells. PGE₂ impaired VD₃‐induced expression of cathelicidin and concomitant activation of autophagy during Mycobacterium tuberculosis (Mtb) infection and facilitated intracellular Mtb growth in human macrophages. An EP4 agonist also significantly promoted Mtb survival in human macrophages. Our results indicate that PGE₂ inhibits hCAP18/LL‐37 expression, especially VD₃‐induced cathelicidin and autophagy, which may reduce host defense against Mtb. Accordingly, antagonists of EP4 may constitute a novel adjunctive therapy in Mtb infection.—Wan, M., Tang, X., Rekha, R. S., Muvva, S. S. V. J. R., Brighenti, S., Agerberth, B., Haeggstro¨m, J. Z. Prostaglandin E₂ suppresses hCAP18/LL‐37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection. FASEB J. 32,2827–2840 (2018). www.fasebj.org