The oxygen isotopes of water (H218O and H216O) are tracers widely used for the investigation of Earth science problems. The tracer applications are based on the premise that the 18O/16O ratio of open-water evaporation (δ18OE) can be calculated from environmental conditions. A long-standing issue concerns the role of kinetic fractionation, or diffusion transport, in the evaporation process. Here we deployed an optical instrument at a large lake (area 2,400 km2) to make in situ measurement of δ18O and δD of atmospheric vapor, then determined δ18O and δD of open-water evaporation using the gradient-diffusion method. Our results show a much weaker kinetic effect than suggested by the kinetic factor εk adopted in some previous studies of lake hydrology (14.2‰). By incorporating into the H218O isotopic mass balance of the lake a lower εk value (about 6.2‰) used for ocean evaporation in global climate models, we obtain an annual lake evaporation rate that agrees with an independent eddy-covariance observation, but the rate is 72% higher than if the commonly used lake εk value of 14.2‰ is applied. The applicability of this results to small lakes is uncertain and in need of field-based assessment.