A rapidly approaching dark object evokes an evolutionarily conserved fear response in both vertebrates and invertebrates, young to old. A looming visual stimulus mimics an approaching object and triggers a similarly robust fear response in mice, resulting in freeze and flight. However, the retinal neural pathway responsible for this innate response has not been fully understood. We first explored a variety of visual stimuli that reliably induced these innate responses, and found that a looming stimulus with two-day acclimation consistently evoked fear responses. Because the fear responses were triggered by the looming stimulus with moving edges, but not by a screen flipping from light to dark, we targeted the starburst amacrine cells (SACs), crucial neurons for retinal motion detection. We utilized intraocular injection of diphtheria toxin (DT) in mutant mice expressing diphtheria toxin receptors (DTR) in SACs. The looming-evoked fear responses disappeared in half of the DT-injected mice, and the other mice still exhibited the fear responses. The optomotor responses were reduced or eliminated, which occurred independent of the disappearance of the fear responses. A histological examination revealed that ON SACs were reduced in both mouse groups preserved or absent fear responses. In contrast, the number of OFF SACs was different among two groups. The OFF SACs were relatively preserved in mice exhibiting continued fear responses, whereas they were ablated in mice lacking fear response to looming stimulation. These results indicate that OFF SACs and the direction-selective pathway in the retina play a role in looming-induced fear behaviors.Significance StatementIn response to a suddenly approaching dark object, mice exhibit a defensive response: either flight to a refuge or freeze in the same spot. How the visual system evokes this response in the mouse has not been fully understood. We focused on the initial neural component of the visual system, the retina, and examined a type of neuron known for sensing object motion: the starburst amacrine cell. We found that ablation of OFF starburst amacrine cells removed the dark object-evoked defensive responses. We believe that our findings will contribute to understanding the neural network connecting the visual system and the brain circuit regarding fear and emotion.