Melatonin is an indole amine that is synthesized and interacts with glycolytic proteins during alcoholic fermentation in Saccharomyces and non-Saccharomyces yeasts depending on their yeast fermentative capacity. Due to its importance as a bioactive compound and antioxidant molecule, the aim of this study was to analyse the intracellular melatonin profile and melatonin-protein interactions in a large number of wine yeast species during alcoholic fermentation and respiration to determine whether these interactions were related to a specific carbon metabolism. Melatonin concentrations were analysed by liquid chromatography‒mass spectrometry, and proteins bound to melatonin were immunopurified at different time points during yeast growth. Intracellular melatonin followed a similar pattern in all yeast species, with a peak in production during the lag phase and low or no melatonin detected in the exponential or stationary phase. However, melatonin was bound only to proteins in good fermentative yeasts, specifically during alcoholic fermentation but not during cellular respiration. The absence of this binding in some non-Saccharomyces yeasts could be related to their poor fermentative capacity. This study establishes for the first time a relationship between carbon metabolism and the interaction of melatonin with proteins in yeast cells, indicating that melatonin might play a regulatory role in glucose metabolism, as observed in human cells.