Mitochondrial Sirtuin 5 (SIRT5) regulates novel protein post-translational modifications on metabolic enzymes by catalyzing the removal of succinyl, malonyl, and glutaryl moieties. SIRT5 represents an attractive therapeutic target, as it is over-expressed in many cancers, and SIRT5 knockdown inhibits the anchorage independent growth of human cancer cells with little effect on normal cells. To examine SIRT5 loss in vivo, we generated SIRT5 wild-type (WT) or knockout (KO) MMTV-PyMT mice, which are prone to mammary adenocarcinomas and lung metastases. SIRT5 KO mice had increased survival, decreased tumor size, and lacked lung metastases, when compared to SIRT5 WT mice. Furthermore, I observed that pharmacological inhibition of SIRT5 impaired mammary tumor growth in both transgenic and human breast cancer xenograft mouse models. In this proposal, I will investigate the molecular mechanism by which SIRT5 promotes tumorigenesis and metastasis. In Aim 1, I will test whether desuccinylation of isocitrate dehydrogenase 2 (IDH2) by SIRT5 promotes breast cancer through reactive oxygen species (ROS) mitigation. In Aim 2, I will investigate whether SIRT5 regulates the tumor microenvironment. Based on prior studies indicating that SIRT5 can modulate immune cell function and my own preliminary data, I hypothesize that SIRT5 could also regulate the tumor niche via suppression of antitumorigenic macrophages. In Aim 3, we will identify SIRT5-regulated substrates in tumor-associated macrophages that are important for promoting cancer progression. Our findings could open a new avenue of targeting cancer cells and the proposed work will provide novel mechanistic insights into the role of SIRT5 in breast cancer progression.