Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.