Understanding the interactive responses of agroecosystem water use efficiency (WUE) to climatic and vegetation factors is significant for revealing how ecohydrological processes vary between the carbon and water cycles in the changing climate. Based on data from 11 eddy covariance flux sites for a total of 32 site-years in four typical agroecosystems, including winter wheat (Triticum aestivum L.), paddy rice (Oryza sativa L.), soybean (Glycine max L. Merr.) and summer maize (Zea mays L.), evapotranspiration (ET) of the four crops was partitioned based on the theory of underlying WUE. Seasonal dynamics of transpiration (T), ET, gross primary production (GPP), T-based WUE at the canopy level (WUEc) and ET-based WUE at the agroecosystem level (WUEe) were investigated. A series of climatic and vegetation factors affecting WUEc and WUEe were quantified using the path analysis method. T/ET was 0.67 ± 0.04 for summer maize, followed by winter wheat, soybean and paddy rice, with values of 0.65 ± 0.07, 0.60 ± 0.05 and 0.57 ± 0.02, respectively. T, ET, GPP and WUEe increased as crops grew but decreased as crops matured. However, WUEc did not change as apparently as WUEe since T and GPP changed synchronously throughout the growing season. Summer maize possessed the largest WUEc of 5.69 ± 0.29 g C kg−1 H2O, followed by winter wheat, soybean and paddy rice, with values of 4.47 ± 0.87, 3.79 ± 0.20 and 2.60 ± 0.17 g C kg−1 H2O, respectively. WUEe followed the same order as that of WUEc among the four croplands, with values of 3.32 ± 0.71, 2.73 ± 0.81, 1.90 ± 0.10 and 1.47 ± 0.09 g C kg−1 H2O, respectively. WUEc was mainly regulated by canopy water consumption than carbon sequestration, while carbon sequestration regulated WUEe more than water consumption at the ecosystem scale. WUEc was mainly controlled by climatic factors (vapor pressure deficit, net radiation, air temperature and soil temperature) in the croplands, while the leaf area index exerted stronger controls on WUEe variations than climatic factors by its strong regulation on T/ET in the four croplands.