This study was aimed at testing the efficacy of a training program involving human values education and mindfulness skills as a tool for emotional self-regulation and stress prevention. Forty school teachers took part in this study. Participants were assigned to two different groups: experimental (N= 25) and control (N= 15). Subjective, behavioral and psychophysiological (respiration and skin conductance) measures were taken. The results revealed significant effects of the training program on the three types of measures. The experimental group showed significant improvements in the subjective measures of negative affect, anxiety, depression, worry, perceived stress, emotional comprehension and muscular pain. Participants who received the training program also showed, in the psychophysiological test, less activation of the sympathetic nervous system associated with less reactivity to unpleasant and stressful situations. In addition, behavioral measures suggested an improvement in the quality of attention. It is concluded that training in human values and mindfulness skills is an effective tool for emotional self-regulation and stress prevention helping to improve the quality of life for teaching professionals.