IF₁ is a natural inhibitor protein for mitochondrial F_oF₁ ATP synthase that blocks catalysis and rotation of the F₁ by deeply inserting its N-terminal helices into F₁. A unique feature of IF₁ is condition-dependent inhibition; although IF₁ inhibits ATP hydrolysis by F₁, IF₁ inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF₁-inhibited bovine mitochondrial F₁ (bMF₁). The results show that IF₁-inhibited F₁ is efficiently activated only when F₁ is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF₁ inhibition. Investigation of mutant IF₁ with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F₁.