Although subordinates’ stress is closely correlated with ambidextrous leadership (AL), surprisingly little organizational research considered the role of challenge-hindrance stressor framework (CHSF) when discussing the leadership approach. We enrich the ambidextrous leadership theory by integrating it with CHSF to argue that AL comprises a double-edged sword for innovative work behavior (IWB) through both the positive mechanism of perceived challenge stressors (PCS) and the negative mechanism of perceived hindrance stressors (PHS). Additionally, we propose the curvilinear moderating role of conscientiousness (CO) drawing upon the resource allocation framework (RAF). We found support for our hypotheses across a video experiment (84 participants across two conditions) and a survey study (three-wave, 218 matched samples). Taken together, our findings provide new evidence for the double-edged sword effect of AL and reveal the importance of examining subordinates’ forms of stress and CO in this leadership process.