The effects of a novel method non-thermal cold plasma (CP) treatment on microbial control, quality maintenance, and γ-aminobutyric acid (GABA) accumulation of shred carrots were investigated. Results revealed CP treatment suppressed microbial growth and inhibited the increase in L* value and whiteness index, while stimulated the generation of reactive oxygen species. Meanwhile, it also enhanced activities and transcriptions of glutamate decarboxylase and GABA transaminase in GABA shunt, and strengthened the polyamine degradation pathway via enhancing activities of diamine oxidase (DAO), polyamine oxidase (PAO), aminoaldehyde dehydrogenase (AMADH), and the expression of DcPAO, thus in turn promoting GABA accumulation through facilitating the conversion from glutamate and polyamines into GABA. Moreover, CP treatment improved the energy status and reducing power by upregulating energy metabolism and pentose phosphate pathway. Therefore, these findings indicated that CP treatment was effective for microbial reduction, quality preservation, and GABA accumulation in fresh-cut carrots, thereby providing potential strategies for the preservation and processing of fresh-cut produce.