Amphotericin B (AmB) is a polyene antifungal drug widely used for systemic fungal infections. In this study, a metabonomic method using gas chromatography-mass spectrometry (GC/MS) was developed to characterize the metabolic profiles of Candida albicans cells exposed to AmB. Thirty-one differentially produced metabolites between AmB-treated and the control groups were identified, among which 10 metabolites were upregulated and 21 metabolites were downregulated. These differentially produced metabolites were mainly involved in polyamines synthesis, tricarboxylic acid (TCA) cycle, oxidative stress, glutathione metabolism, lipid synthesis and glycolysis. Further experiments showed that the polyamines including putrescine, spermidine, and spermine played an important role in the sensitivity of C. albicans cells upon AmB treatment, and combined use of AmB and inhibitors of polyamine biosynthesis pathway might be a potential antifungal strategy. This study provided a systemic view of the metabolic pattern in C. albicans upon exposure to AmB, which shed new light on the mechanisms of action of antifungal agents.