Nicotine, like several other abused drugs, is known to act on the reward system in the brain. Smoking-associated cues produce smoking urges and cravings accompanied by autonomic dysfunction to these cues in smokers. The present study was aimed at investigating whether cues related to smoking elicit the autonomic response in smokers. The subjective and physiological reactivity of 7 smokers and 12 nonsmokers in a supine position to smoking-related visual cues was assessed under indirect dim light using a self-assessment manikin and a specially designed pupillometer. The experimental procedure consisted of the elicitation and measurement of pupil size (PS) while the subjects viewed a smoking image and images from three valence-defined categories (i.e., pleasant, unpleasant, and neutral), based on normative affective ratings selected from the International Affective Picture System. Both groups produced significantly larger PS increases in response to pleasant or unpleasant images compared to neutral images. Smokers, viewing smoking-related visual cues but no other affective images, produced significantly larger PS's compared to nonsmokers. Moreover, smokers rated the smoking image with more pleasure and arousal than nonsmokers. These findings suggest that cues related to smoking induce not only a subjective emotional alteration, but also sympathetic activation, measured by the time-series PS data in smokers.