Ambidexterity organization, which is defined as the ability of an organization to simultaneously pursues exploration and exploitation, has received attention by researchers who have examined its beneficial effect on organizational performance and success. This study attempted to examine the positive effect of ambidextrous organization culture (AOC), which is regarded as the core characteristic of ambidextrous organizations by using a multilevel model. Specifically, this study examined the effects of AOC on members’ job performance and the mediating role of psychological capital in the relationship between AOC and job performance. The results indicated that AOC had a significantly positive relationship with job performance even after controlling various organizational and individual variables. Moreover, we found that psychological capital fully mediated the relationship between AOC and members’ job performance. This study provides theoretical contributions by empirically examining the positive effect and mechanism of AOC. Furthermore, this study offers practical implications in how practitioners can manage their organizational culture, by helping shape the direction of organizational culture management.