3-Methylglutaconic (3MGC) aciduria occurs in numerous inborn errors associated with compromised mitochondrial energy metabolism. In these disorders, 3MGC CoA is produced de novo from acetyl CoA in three steps with the final reaction catalysed by 3MGC CoA hydratase (AUH). In in vitro assays, whereas recombinant AUH dehydrated 3-hydroxy-3-methylglutaryl (HMG) CoA to 3MGC CoA, free CoA was also produced. Although HMG CoA is known to undergo non-enzymatic intramolecular cyclisation, forming HMG anhydride and free CoA, the amount of free CoA generated increased when AUH was present. To test the hypothesis that the AUH-dependent increase in CoA production is caused by intramolecular cyclisation of 3MGC CoA, gas chromatography-mass spectrometry analysis of organic acids was performed. In the absence of AUH, HMG CoA was converted to HMG acid while, in the presence of AUH, 3MGC acid was also detected. To determine which 3MGC acid diastereomer was formed, immunoblot assays were conducted with 3MGCylated BSA. In competition experiments, when α-3MGC IgG was preincubated with trans-3MGC acid or cis-3MGC acid, the cis diastereomer inhibited antibody binding to 3MGCylated BSA. When an AUH assay product mix served as competitor, α-3MGC IgG binding to 3MGCylated BSA was also inhibited, indicating cis-3MGC acid is produced in incubations of AUH and HMG CoA. Thus, non-enzymatic isomerisation of trans-3MGC CoA drives AUH-dependent HMG CoA dehydration and explains the occurrence of cis-3MGC acid in urine of subjects with 3MGC aciduria. Furthermore, the ability of cis-3MGC anhydride to non-enzymatically acylate protein substrates may have deleterious pathophysiological consequences.