The effect of defects on electron-hole separation is not always clear and is sometimes contradictory. Herein, we initially built clear models of two-dimensional atomic layers with tunable defect concentrations, and hence directly disclose the defect type and distribution at atomic level. As a prototype, defective one-unit-cell ZnIn2S4 atomic layers are successfully synthesized for the first time. Aberration-corrected scanning transmission electron microscopy directly manifests their distinct zinc vacancy concentrations, confirmed by positron annihilation spectrometry and electron spin resonance analysis. Density-functional calculations reveal that the presence of zinc vacancies ensures higher charge density and efficient carrier transport, verified by ultrafast photogenerated electron transfer time of similar to 15 ps from the conduction band of ZnIn2S4 to the trap states. Ultrafast transient absorption spectroscopy manifests the higher zinc vacancy concentration that allows for similar to 1.7-fold increase in average recovery lifetime, confirmed by surface photovoltage spectroscopy and PL spectroscopy analysis, which ensures promoted carrier separation rates. As a result, the one-unit-cell ZnIn2S4 layers with rich zinc vacancies exhibit a carbon monoxide formation rate of 33.2 mu mol g(-1) h(-1), roughly 3.6 times higher than that of the one-unit-cell ZnIn2S4 layers with poor zinc vacancies, while the formers photocatalytic activity shows negligible loss after 24 h photocatalysis. This present work uncovers the role of defects in affecting electron-hole separation at atomic level, opening new opportunities for achieving highly efficient solar CO2 reduction performances.