In order to reveal the role of phytoplankton in the spatio-temporal distribution of alkaline phosphatase activity (APA), monthly investigations were conducted in the Xiaojiang River, a tributary of the Three Gorges Reservoir in China. Different APA fractions, environmental parameters, and phytoplankton communities were followed. High spatio-temporal variations of APA were observed, with the highest value in summer and the lowest in winter. The annual average APAT (total alkaline phosphatase activity) ranged from 7.78-14.03 nmol∙L-1∙min-1 with the highest in the midstream and the lowest in the estuary. The dominant phytoplankton phyla in summer and winter were Cyanophyta and Bacillariophyta, respectively. The mean cell density in the midstream and in the estuary was 5.2 × 107 cell∙L-1 and 1.4 × 107 cell∙L-1, respectively. That APA>3.0 μm was significantly higher than APA0.45-3 μm indicating phytoplankton was the main contributor to alkaline phosphatase. Correlation analysis indicated the dominant species and cell density could determine the distribution pattern of APA. Turbidity, total phosphorus, chemical oxygen demand, water temperature (WT), pH and chlorophyll a were proved to be positively correlated with APA; soluble reactive phosphorus, conductivity, transparency and water level(WL) were negatively correlated with APA. It was concluded that spatio-temporal heterogeneity of APA determined by phytoplankton communities was related to WT and WL.