Background: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid β-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. Methods: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. Results: Itaconate reacts with the CAC causing irreversible inhibition. Dose–response experiment performed with the native and recombinant protein showed IC50for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.