Men who misuse alcohol tend to experience negative affect, which may entail difficulties in regulating emotions to cope effectively with stressful or anxiety-provoking situations, thus increasing the risk of alcohol relapse. This dysphoric state has been associated with alexithymia, which compromises an individual’s abilities to acknowledge, recognize, and regulate emotional states. A physiological correlate of emotional regulation is autonomic flexibility, as shown by emotional dysregulation in men who misuse alcohol being correlated with reduced parasympathetic activation to control heart rate variability during stress and/or conflict situations. Hence, the main aim of this study was to investigate whether long-term abstinent alcoholic (LTAA) men exhibit higher levels of negative affect and sympathetic activation (cardiovascular and electrodermal) in response to acute standardized laboratory stress than non-alcoholic controls. In addition, we hypothesized that the higher the alexithymic traits, the greater would be the increase in negative affect and sympathetic activation in response to stress, especially in LTAAs. Our data demonstrated that LTAAs experienced slightly greater increases in anxiety, states of anger, and worsening of mood than controls. Moreover, they exhibited lower high-frequency heart rate variability, respiratory sinus arrhythmia values, shorter pre-ejection periods, and higher respiratory rates than controls. Finally, alexithymic traits imply greater worsening of mood and sympathetic predominance (shorter pre-ejection periods and smaller magnitude of response), with the associations being stronger in LTAAs. These findings indicate a different emotional and cardiovascular response to psychosocial stress in LTAA than non-alcoholic men. Improving our knowledge of the way this population reacts to stress may help identify risk factors for alcohol relapse.