In psychiatric nursing, female nurses tend to spend more time building rapport with patients and developing cooperative working relationships with colleagues; they encounter more sexual harassment by patients. In contrast, male nurses respond to aggressive patients and tend to resist physically caring for female patients; they encounter more physical and verbal assault from patients. These gender differences might result in differences in job-related stress. We quantitatively examined gender differences in psychiatric nurses' job stress. The Psychiatric Nurse Job Stressor Scale and the Stress Reaction Scale of the Brief Job Stress Questionnaire were administered to 159 female and 85 male Japanese psychiatric nurses. The results indicated that female nurses had significantly higher stress levels than males related to psychiatric nursing ability, attitude towards nursing, and stress reactions of fatigue and anxiety. Moreover, the factors affecting stress reactions differed somewhat between sexes. In particular, male nurses reported that greater irritability was affected by patients' attitudes. Their anxiety and somatic symptoms were affected by their attitude towards nursing, and depressed mood was affected by psychiatric nursing ability. Knowledge of these differences can lead to better mental health-care interventions for psychiatric nurses.