AbstractNicotine‐associated cues can trigger reinstatement in humans as well as in animal models of drug addiction. To date, no behavioral intervention or pharmacological treatment has been effective in preventing relapse in the long term. A large body of evidence indicates that N‐acetylcysteine (N‐AC) blunts the activation of glutamatergic (GLUergic) neurons in the nucleus accumbens (Nacc) associated with reinstatement. We evaluated the effect of an experimental cue exposure therapy (eCET) alone or in combination with N‐AC to verify whether restoring GLU homeostasis enhances extinction of nicotine‐associated cues. Rats were trained to associate discriminative stimuli with intravenous nicotine or saline self‐administration. Reinforced response was followed by cue signals. After rats met the self‐administration criteria, the lasting anti‐relapse activity of i.p. N‐AC or vehicle was assessed in three different experimental conditions after 14 days of treatment: treatment + eCET; treatment + lever‐presses extinction (LP‐EXT); and treatment + abstinence. N‐AC 100 mg/kg, but not 60 mg/kg, induced anti‐relapse activity that persisted 50 days after treatment only when paired with either LP‐EXT or eCET with the greater activity found in the latter condition. To identify potential mechanisms for behavioral results, separate groups of rats that received either N‐AC or vehicle + eCET were killed at different time points for Nacc Western‐blot analysis. Seven days after treatment, chronic N‐AC restored the expression of proteins crucial for GLU homeostasis, while at 50 days, it increased the expression of type II metabotropic GLU receptors. These results suggest that N‐AC treatment in combination with eCET may offer a novel strategy to prevent relapse in nicotine addiction.