Single-layer molybdenum disulfide (SLMoS2) are applied as a hot 2D nanosheet in various fields involving water treatments. Both intentional design and environmental or biological processes induce many nanoholes in SLMoS2. However, the effects of nanoholes on the environmental stability and ecotoxicity of SLMoS2 remain largely unknown. The present work discovered that visible-light irradiation induced nanoholes (diameters, approximately 20 nm) in the plane of SLMoS2, with irregular edges and increased interplanar crystal spacing. The ratios of Mo to S in pristine and transformed SLMoS2 were 0.53 and 0.33, respectively. After 96 h exposure at concentrations from 0.1 to 1 mg/L, the above nanoholes promoted algal division, induced a stress-response hormesis, decreased the generation of •OH, and mitigated the cell shrinkage and wall rupture of Chlorella vulgaris induced by SLMoS2. In terms of stress response, the nanohole-bearing SLMoS2 induced fewer vacuoles and polyphosphate bodies of Chlorella vulgaris than the pristine form. Metabolomic analysis revealed that nanoholes perturbed the metabolisms of energy, carbohydrates, and fatty acids. This work proposes that nanoholes cause obvious effects on the environmental fate and ecotoxicity of SLMoS2 and that the environmental risks of engineered nanomaterials should be reevaluated using nanohole-bearing rather than pristine forms for testing.