These authors contributed equally to this work.Keywords: antimicrobial peptides, cathelicidin, innate immunity, P2RX7, tuberculosis, vitamin D Abbreviations: 1,25(OH) 2 D 3 , 1,25-dihydroxy vitamin D 3 ; AMPK, adenosine monophosphate-activated protein kinase; AMPs, antimicrobial peptides; ATG, autophagy related; BECN1, Beclin 1, autophagy related; CAMP, cathelicidin antimicrobial peptide; CFUs, colony-forming units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MDMs, monocyte-derived macrophages; Mtb, Mycobacterium tuberculosis; P2RX7, purinergic receptor P2X, ligand gated ion channel, 7; PBA, 4-phenylbutyrate; PBS, phosphate-buffered saline; PtdIns3K, phosphatidylinositol 3-kinase; RNA18S/18S rRNA, RNA 18S ribosomal; SQSTM1, sequestosome 1; TB, tuberculosis LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D 3 (1,25[OH] 2 D 3 ), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca 2C , and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.