Activated immune cells display a phenotype of accelerated metabolism with enhanced glycolysis and a disruption of the TCA cycle, ultimately causing the accumulation of metabolites. Succinate can then serve as a signalling molecule or as a post-translational modification of lysine residues. The two cytokines RANKL and M-CSF differentiate macrophages into bone resorbing osteoclasts, a process which is characterized by changes in metaboblic activity to allow for energy consuming processes like cell fusion or bone resorption. We had previously observed that succinate treatment increased the number of TRAP positive osteoclasts. High levels of succinate can be detected in the synovial fluid of rheumatoid arthritis patients, but the importance of protein succinylation for the disease is unknown.To identify succinylated proteins under osteoclastogenic conditions, we used an immunoaffinity-based LC-MS/MS analysis combined with dimethyl-labelling. Most lysine succinylated proteins were mitochondrial and related to metabolic activities. The proteomic approach implies that protein succinylation could play a role in the regulation of mitochondrial activities during osteoclast differentiation. We identified citrate synthase (CS), the enzyme that starts the TCA cycle, ATP synthase of complex V (ATP5B), the protein that generates ATP in the TCA cycle and serine hydroxymethyltransferase-2 (SHMT2), an enzyme required for redox homeostasis as potentially interesting. Co-immunoprecipitation assays validated all three proteins as targets of lysine succinylation upon RANKL stimulation. CS was the only protein where succinylation was only detected after RANKL stimulation. Using whole cell extracts, we observed that RANKL treatment increased CS activity compared to unstimulated control cells. For ATP5B, we identified two succinyl-lysine sites that had previously been reported for acetylation. As acetylated ATP5B decreases ATP production and ATP5B-deficient mice fail to differentiate osteoclasts due to impaired mitochondrial activity, our results imply that RANKL-induced ATP5B succinylation could be a critical factor in the context of energy production during osteoclastogenesis.