Most studies in the project management field emphasized the effects of leaders or managers, but these effects have rarely been examined at the project level. To cover this research gap, this study applies organizational ambidexterity theory to examine the effects among ambidextrous leadership, ambidextrous culture and sustainability-based project performance. Using a valid sample of 217 project leaders and members from Chinese construction projects, the method of multiple linear regression was adopted to assess the direct relationship among ambidextrous leadership, ambidextrous culture and project performance. Moreover, the bootstrapping technique through structural equation modeling, has been used to analyze the mediating effect of ambidextrous culture. Additionally, the sample data was divided into different groups according to the median value of the variables to conduct the ANOVA and to assess the within-group differences. The results indicated a positive and direct relationship that ambidextrous leadership has on project performance and ambidextrous culture. In addition, there was also a mediating impact of ambidextrous leadership on project performance via ambidextrous culture. Thus, ambidextrous leadership combined with transformational leadership as well as transactional leadership likely has a stronger positive impact on project performance through fostering the adaptive culture and consistent culture. Our findings contribute to an in-depth understanding of the role of the leader and culture for project outcomes. The project-based organization in construction projects could train project leaders’ ambidextrous leadership behavior to facilitate the formation of an ambidextrous culture and to increase project performance. Moreover, this study enriches the existing literature on leadership and project management by highlighting the important path of ambidextrous leadership and ambidextrous culture on the performance at the project level and going beyond the single leadership behavior or single culture.