Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. Thioredoxin-1 (Trx-1) is an important regulator of neuroprotection, and inhibits morphine-induced hyperlocomotion, reward and withdrawal signs, as well as blocks methamphetamine (METH)-induced conditioned place preference (CPP). The nucleus accumbens (NAc) is essential for relapse like behavior in reinstatement animal models. In the present study, we aimed to investigate the role of Trx-1 in the NAc in METH-primed reinstatement by using a reinstatement procedure in mice. Adeno-associated virus vectors expressing shRNA-mTrx-1 (AAV-shRNA-mTrx-1) were bilaterally microinjected into the NAc after METH-CPP extinction. The results showed that Trx-1 downregulation in the NAc promoted the reinstatement of METH-CPP. We also examined the expression of N-methyl-D-asparate (NMDA) receptor 2B subunit (GluN2b), the levels of phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated cAMP-response element binding protein (p-CREB) in the NAc by western blot analysis, and found that the GluN2b expression, p-ERK and p-CREB levels were increased in the NAc in response to low-dose METH in AAV-shRNA-mTrx-1 mice, but were not changed in control and AAV-vehicle mice. These data indicate that the increased GluN2b expression, and p-ERK and p-CREB levels in the NAc of AAV-shRNA-mTrx-1 mice may be responsible for the METH-primed reinstatement. Thus, we suggest that downregulation of Trx-1 in the NAc may make mice more sensitive to METH reinstatement.