An increasing number of studies have demonstrated that humor can impact interpersonal relationships in organizations and employee well-being. However, there is little evidence coming from intervention studies in organizational settings. In response, we developed a training following the principles of positive psychology that aims at improving employees’ adaptive use of humor as a successful mechanism to deal with stress. In this study, we assess the effectiveness of such training and its impact on employee well-being. Results from this one-group intervention study in an emergency ambulance service (N = 58) revealed that the participants reported higher levels of cheerfulness (Z = −3.93; p < 0.001) and lower levels of seriousness (Z = −3.32; p < 0.001) after being exposed to the training. Indeed, the participants reported lower scores on psychological distress after the training (Z = −3.35; p < 0.001). The effect size of the training was medium (r = 0.31 to 0.36), suggesting that interventions to improve adaptive humor at work can be a useful resource to deal with workplace stress and foster employee well-being. These results may have interesting implications for designing and implementing positive interventions as well as for developing healthy organizations.