Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the Mitochondrial Calcium Uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca²⁺ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analysed from several models of obese and diabetic rodents and in obese patients, as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca²⁺]_mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and ROS formation. Finally, our data suggest a strong relationship between [Ca²⁺]_mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes, and may be associated with the differential metabolic profiles of VAT and SAT.