Research on strategic human resource (HR) management and organizational ambidexterity has assumed that organizational ambidexterity originates from operational managers that pursue both exploratory and exploitative activities. Yet, multilevel insights are absent about how and through which mechanisms HR practices may actually facilitate operational manager ambidexterity and how their ambidexterity may result into organizational ambidexterity. Our multisource and multilevel data from 467 operational managers and 104 senior managers within 52 firms reveals that the top-down effects of ability- and motivation-enhancing HR practices on operational manager ambidexterity are partially mediated by their role breadth self-efficacy and intrinsic motivational orientation. Furthermore, we find that the bottom-up relationship between operational manager and organizational ambidexterity is contingent on firm opportunity-enhancing HR practices. With that, our study provides important new multilevel insights into the effectiveness of strategic HR systems in supporting individual and organizational ambidexterity.