Background <p>Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by aberrant fibroblast activation and extracellular matrix deposition. Emerging evidence implicates mitochondrial dysfunction in IPF pathogenesis. Although CAMKK2 has been implicated in mitochondrial function associated with diabetic nephropathy, its role and underlying mechanisms in IPF remain unclear. This study aims to investigate the role of CAMKK2 in IPF.</p> Methods <p>This study employed AAV-CAMKK2 mice, primary human/mouse lung fibroblasts, MRC-5 cells, and IPF patient samples. The CAMKK2 inhibitor (STO-609), shCAMKK2 and Flag-CAMKK2 overexpression plasmid were used to investigate the role of CAMKK2 in lung fibroblasts. Fibroblast activity was assessed by transwell migration, collagen contraction, and wound-healing assays. Mitochondrial function (ROS, mitochondrial membrane potential (MMP), ATP and oxygen consumption rate (OCR)) was measured by ROS assay, JC-1 assay, ATP assay and Seahorse assay. Mitochondrial dynamics (MFN1/MFN2, DRP1), PGC-1α, and p-^T172AMPK were analyzed alongside fibrotic markers (FN1, COL1A1 and α-SMA) by Western blotting and RT-qPCR. The AMPK inhibitor (Compound C) and PGC-1α inhibitor (SR-18292) were used to investigate the role of AMPK and PGC-1α in this study.</p> Results <p>This study revealed that CAMKK2 expression was significantly downregulated in pulmonary fibrosis, concomitant with impaired mitochondrial function-related proteins (PGC-1α, MFN1and MFN2). In vitro experiments demonstrated that CAMKK2 inhibition or shCAMKK2 knockdown exacerbated fibroblast activation and extracellular matrix (ECM) production in both MRC-5 cells and primary mouse lung fibroblasts. Conversely, CAMKK2 overexpression attenuated TGF-β1-induced fibroblast activation and ECM deposition in MRC-5 cells and primary human lung fibroblasts. Further investigation established that CAMKK2 overexpression normalized mitochondrial morphology, enhanced MMP, ATP content and respiratory capacity to ameliorate mitochondrial dysfunction in TGF-β1–induced MRC-5 cells. Moreover, CAMKK2 upregulated mitochondrial fusion protein expression (MFN1 and MFN2) and suppressed fission (DRP1) in MRC-5 cells. Mechanistically, CAMKK2 regulated mitochondrial dynamics and OXPHOS function via the AMPK/PGC-1α pathway in TGF-β1–induced MRC-5 cells, as evidenced by rescued protein expression (MFN1 and MFN2), and reversal of these effects following AMPK or PGC-1α inhibition. In vivo studies showed that AAV-mediated CAMKK2 delivery significantly attenuated bleomycin-induced pulmonary fibrosis in mice.</p> Conclusions <p>In summary, these findings collectively demonstrate that CAMKK2 regulated mitochondrial dynamics and OXPHOS function via AMPK/PGC-1α signaling pathway to alleviate pulmonary fibrosis in lung fibroblasts. Therefore, targeting CAMKK2 presents a novel and promising therapeutic strategy for the treatment of pulmonary fibrosis.</p>