Exposure to repetitive pain procedures in the neonatal intensive care unit (NICU) leads to long-term effects on pain response in adults. However, the underlying mechanisms in the central nervous system (CNS) are unclear. This study aimed to invesitgate the role of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) in the nucleus accumbens (NAc), including increased protein expression, cell number, and specific enzyme activity, which helps accelerate pain. We found that repetitive pain induced the upregulation of stargazin, but there was no significant difference in N-ethylmaleimide sensitive factor (NSF). S-nitrosylation was examined using biochemical assays. However, the NO-induced S-nitrosylation levels of stargazin and NSF in the NAc of neonatal rats in the Needle group were significantly increased. Moreover, gephyrin expression was downregulated, while that of Na