This article aims to examine the relationship between ambidextrous organisational cultures and ambidextrous learning by higher education institutions, and to scrutinise how the ambidextrous leadership and knowledge of these institutions relate to their ambidextrous cultures. Survey data from 474 university administrators within the Gulf Cooperation Council region, serve as the basis for empirical insights based on structural equation modelling. Survey responses show significant and positive correlation between ambidextrous organisational culture and ambidextrous learning, and additionally reveal that ambidextrous knowledge and ambidextrous leadership significantly and positively relate to ambidextrous organisational cultures. By including ambidextrous forms of culture, leadership, and knowledge constructs to explain ambidextrous learning by organisations, this study makes contributions to organisational ambidexterity theory. Theoretically, this article adds to understanding on the determinants of ambidextrous learning by providing evidence of a culture-learning praxis, while managerially, the article urges for ambidextrous culture-oriented approaches to academic staff development.