The effect of exogenous γ-aminobutyric acid (GABA) and GABA synthesis inhibitor 3-mercaptopropionic acid (3-MP) on GABA metabolism and quality in fresh-cut pumpkin was investigated, and the possible mechanism was studied. The results indicated that exogenous GABA induced the accumulation of endogenous GABA and promoted the conversion of glutamic acid to GABA in fresh-cut pumpkin through the catalytic conversion of highly active glutamate decarboxylase (GAD). The 3-MP-treated pumpkin retained lower GABA content than the control, and the activities of GAD and GABA transaminase (GABA-T) were inhibited. The GABA-treated pumpkins possessed relatively lower polyamines (putrescine, spermine and spermidine) contents in comparison with the control during storage. 3-MP treatment restrained the activities of polyamine oxidase (PAO), diamine oxidase (DAO) and amino aldehyde decarboxylase (AMADH). These results demonstrated that GABA treatment could activate the polyamine degradation pathway and promote GABA enrichment in pumpkin. Meanwhile, no significant differences in maximum colony count, color, β-carotene content and soluble solids content were observed among treatments during storage, which suggested that exogenous GABA had little effect on the quality of fresh-cut pumpkin.