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BACKGROUND: Carnosic acid, a bioactive compound found in rosemary, has been shown to reduce inflammation and reactive oxygen species (ROS). However, its mechanism of action in osteoclast differentiation remains unclear. OBJECTIVE: To investigate the effects of carnosic acid on osteoclast activation, ROS production, and mitochondrial function. METHODS: Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro. Different concentrations of carnosic acid (0, 10, 15, 20, 25 and 30 μmol/L) were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration. Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days. The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining, F-actin staining, H2DCFDA probe and mitochondrial ROS, and Mito-Tracker fluorescence detection. Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton, with the highest inhibitory effect observed in the high concentration group (30 μmol/L). Carnosic acid exhibited the most significant inhibitory effect during the early stages (days 1-3) of osteoclast differentiation compared to other intervention periods. Fluorescence imaging using the H2DCFDA probe, mitochondrial ROS, and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential, thereby influencing mitochondrial function. The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1, CTSK, MMP9, and C-fos proteins associated with osteoclast differentiation, and downregulate the expression of NFATc1, Atp6vod2, ACP5, CTSK, and C-fos genes related to osteoclast differentiation. Furthermore, carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation. Overall, carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages. [ABSTRACT FROM AUTHOR]