There are mixed results on the effects of opioid antagonists on acute nicotine response in humans. The present study examined the effects of a single dose of 50 mg oral naltrexone relative to placebo on smoking response in 22 chronic smokers during short-term nicotine abstinence, after acute smoking and subsequent smoking deprivation, and on smoking behavior in a choice paradigm. The results showed that naltrexone significantly reduced immediate postcigarette ratings of smoking craving and desire to smoke and increased light-headedness, dizziness, and head rush (ps < 0.05). Reductions in craving and smoking desire persisted during a subsequent 1 h nonsmoking interval. Naltrexone also was found to significantly reduce the total number of cigarettes smoked in the choice interval, which was supported by objective measures of both reduced CO and plasma nicotine levels (ps < 0.01). Exploratory analyses on potential individual difference factors revealed that smokers with the highest levels of craving during abstinence showed the most pronounced naltrexone attenuation of smoking response. The results support the continued exploration of naltrexone as an adjunct to smoking cessation, especially in identified smoker subgroups most sensitive to the effects of opioid antagonism.