Purpose: We examined the interaction effect of job insecurity (JI) and role ambiguity (RA) on psychological distress in Japanese employees. Methods: Overall, 2184 male and 805 female employees from two factories of a manufacturing company in Japan completed a self-administered questionnaire comprising the scales measuring JI (Job Content Questionnaire), RA (National Institute for Occupational Safety and Health Generic Job Stress Questionnaire), psychological distress (K6 scale), and potential confounders (i.e., age, education, family size, occupational class, and work shift). Taking psychological distress as a dependent variable, hierarchical multiple regression analyses were conducted by gender and employment status (i.e., permanent and non-permanent employees). An interaction term of JI × RA was included in the model. Results: After adjusting for potential confounders, the main effects of JI and RA on psychological distress were significant regardless of gender or employment status. Furthermore, the significant interaction effect of JI × RA on psychological distress was observed among permanent male employees (β = 0.053, p = 0.010). Post hoc simple slope analyses showed that the simple slope of JI was greater at higher levels of RA (i.e., one standard deviation [SD] above the mean) (β = 0.300, p < 0.001) compared to lower levels of RA (i.e., one SD below the mean) (β = 0.212, p < 0.001). On the other hand, the interaction effect of JI × RA was not significant among permanent or non-permanent female employees. Conclusions: The present study suggests that higher levels of RA strengthen the association of JI with psychological distress, at least among Japanese permanent male employees.