The benefits of exercise for smokers attempting to quit are partially dependent on physical activity levels prior to cessation. Mood disturbances can manifest within 30 min of smoking a cigarette and contribute to negative reinforcement of smoking behavior over time, whereas a single bout of aerobic exercise can reduce negative mood states and improve affect on a similar timescale in non-smokers. The acute effects of exercise among non-abstaining smokers immediately after a cigarette are unknown but may have clinical implications for smokers considering cessation. The aim of this study was to examine the concurrent effects of exercise on mood disturbance and prefrontal brain hemodynamic response to emotionally arousing scenes 30 min after smoking a cigarette and to test whether prefrontal brain hemodynamic response was correlated with affective ratings of the scenes. Fifteen cigarette smokers, averaging 47.6 cigarettes/week in the year prior to screening, served as participants. Functional near-infrared spectroscopy was used to measure hemodynamic status over the dorsolateral prefrontal cortex (DLPFC). Participants viewed pleasant, unpleasant, and neutral scenes prior to smoking a cigarette and 30 min later after seated rest or exercise at a preferred intensity. Mood disturbance was assessed by the Profile of Mood States-Brief Form four times: before smoking a cigarette, before and after exercise and rest, and after a post-condition (exercise/rest) exposure to emotionally arousing scenes. Compared to seated rest, cycling after a cigarette reduced mood disturbance (p = =.038) and DLPFC hemodynamic response to unpleasant (p = =.003) and pleasant (p = =.021) scenes relative to neutral scenes. DLPFC hemodynamic response was not related to affective ratings of scenes. We report that cycling for 20 min at a preferred intensity reduces mood disturbances which occur shortly after smoking a cigarette and blunts DLPFC hemodynamic response to emotionally arousing scenes. The findings encourage further investigation of exercise for smokers in the maintenance stage of smoking cessation.