The regulatory focus of individuals tends to influence ambidexterity pursuits; however, the underlying mechanism has remained unclear. This study investigates the role of regulatory focus induced by promotion- and prevention-framed messages for ambidexterity pursuits. We conducted three experiments with above middle-level managers in the new technology innovation context. The results indicate that managers with situationally induced (SI) promotion (prevention) focus tend to have higher opportunity (threat) appraisal (Experiment 1). Opportunity appraisal (but not threat appraisal) mediates the relationship between SI regulatory focus and ambidexterity pursuits (Experiment 2). Organizational risk-taking strategy moderates the links from SI regulatory focus to opportunity appraisal to ambidexterity (Experiment 3). This study provides new insights on the role of regulatory focus for ambidexterity pursuit. It theoretically contributes to the understanding of the mechanisms of how managers can have different cognitive processes in the ambidexterity pursuits when they are responding to the changes in technological environments.