The incubation phenomenon, cue-induced drug craving progressively increasing over prolonged withdrawal, accounts for persistent relapse, leading to a dilemma in the treatment of cocaine addiction. The role of neuronal ensembles activated by initial cocaine experience in the incubation phenomenon was unclear. In this study, with cocaine self-administration (SA) models, we found that neuronal ensembles in the nucleus accumbens shell (NAcSh) showed increasing activation induced by cue-induced drug-seeking after 30-day withdrawal. Inhibition or activation of NAcSh cocaine-ensembles suppressed or promoted craving for cocaine, demonstrating a critical role of NAcSh cocaine-ensembles in incubation for cocaine craving. NAcSh cocaine-ensembles showed a specific increase of membrane excitability and a decrease of inward rectifying channels Kir_2.1 currents after 30-day withdrawal. Overexpression of Kir_2.1 in NAcSh cocaine-ensembles restored neuronal membrane excitability and suppressed cue-induced drug-seeking after 30-day withdrawal. Expression of dominant-negative Kir_2.1 in NAcSh cocaine-ensembles enhanced neuronal membrane excitability and accelerated incubation of cocaine craving. Our results provide a cellular mechanism that the downregulation of Kir_2.1 functions in NAcSh cocaine-ensembles induced by prolonged withdrawal mediates the enhancement of ensemble membrane excitability, leading to incubation of cocaine craving.