SIRT3 (sirtuin 3), a mitochondrial protein deacetylase, maintains respiratory function, but its role in the regulation of innate immune defense is largely unknown. Herein, we show that SIRT3 coordinates mitochondrial function and macroautophagy/autophagy activation to promote anti-mycobacterial responses through PPARA (peroxisome proliferator activated receptor alpha). SIRT3 deficiency enhanced inflammatory responses and mitochondrial dysfunction, leading to defective host defense and pathological inflammation during mycobacterial infection. Antibody-mediated depletion of polymorphonuclear neutrophils significantly increased protection against mycobacterial infection in sirt3 mice. In addition, mitochondrial oxidative stress promoted excessive inflammation induced by Mycobacterium tuberculosis infection in sirt3 macrophages. Notably, SIRT3 was essential for the enhancement of PPARA, a key regulator of mitochondrial homeostasis and autophagy activation in the context of infection. Importantly, overexpression of either PPARA or TFEB (transcription factor EB) in sirt3 macrophages recovered antimicrobial activity through autophagy activation. Furthermore, pharmacological activation of SIRT3 enhanced antibacterial autophagy and functional mitochondrial pools during mycobacterial infection. Finally, the levels of SIRT3 and PPARA were downregulated and inversely correlated with TNF (tumor necrosis factor) levels in peripheral blood mononuclear cells from tuberculosis patients. Collectively, these data demonstrate a previously unappreciated function of SIRT3 in orchestrating mitochondrial and autophagic functions to promote antimycobacterial responses. Abbreviations: Ab: antibody; BCG: M. bovis Bacillus Calmette–Guérin; Baf-A: bafilomycin A; BMDMs: bone marrow-derived macrophages; CFU: colony forming unit; CXCL5: C-X-C motif chemokine ligand 5; EGFP: enhanced green fluorescent protein; ERFP: enhanced red fluorescent protein; FOXO3: forkhead box O3; HC: healthy controls; H&E: haematoxylin and eosin; HKL: honokiol; IHC: immunohistochemistry; IL1B: interleukin 1 beta; IL6: interleukin 6; IL12B: interleukin 12B; MDMs: monocyte-derived macrophages; MMP: mitochondrial membrane potential; Mtb: Mycobacterium tuberculosis; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PMN: polymorphonuclear neutrophil; PPARA: peroxisome proliferator activated receptor alpha; ROS: reactive oxygen species; SIRT3: sirtuin 3; TB: tuberculosis; TEM: transmission electron microscopy; TFEB: transcription factor EB; TNF: tumor necrosis factor.