A comparative metabolomic analysis was conducted on Saccharomyces cerevisiae cells with and without patulin treatment using gas chromatography-mass spectrometry-based approach. A total of 72 metabolites were detected and compared, including 16 amino acids, 29 organic acids and alcohols, 19 sugars and sugar alcohols, 2 nucleotides, and 6 miscellaneous compounds. Principle component analysis showed a clear separation of metabolome between the cells with and without patulin treatment, and most of the identified metabolites contributed to the separation. A close examination of the identified metabolites showed an increased level of most of the free amino acids, an increased level of the intermediates in the tricarboxylic acid cycle, a higher amount of glycerol, a changed fatty acid composition, and a decreased level of cysteine and glutathione in the cells with patulin treatment. This finding indicated a slower protein synthesis rate and induced oxidative stress in the cells with patulin treatment, and provided new insights into the effect of toxic chemicals on the metabolism of organisms.