Background: Gratitude is a general state of thankfulness and appreciation. The majority of empirical studies indicate that there is an association between gratitude and a sense of overall well being..Currently, mental health is especially important in some occupations with harsh work conditions which require strong personal and mental capacities. Psychological well-being and happiness is especially important in personnel working in the healthcare sector. This study aimed to assess whether gratitude training affects psychological well-being and happiness in hospital personnel. Method: In this semi-experimental study with pre-test and post-test, 72 personnel working in 5 hospitals affiliated to Shiraz University of Medical Sciences, Shiraz, Iran, participated. The participants were randomly divided into intervention and control groups using block randomization. Then, the participants in the intervention group were given 10 group sessions of 90 minutes gratitude training. Ryff’s Scale of Psychological Well Being, Subjective Happiness Scale, Gratitude Questionnaire-6, and a Gratitude training package were used for data collection. Results: The results show that there is no significant difference in the mean scores of psychological well-being, domains of psychological well-being, and happiness between the two groups at baseline, and gratitude training significantly affected all domains of psychological well-being (except for autonomy) and happiness. Conclusion: This study demonstrates that Gratitude training is effective in enhancing psychological well-being and Happiness.