Climate change and intensified human activities are regarded as the two driving factors for most river systems having lost their connection with the Tarim River. The Hotan River is the second largest headstream of the Tarim River; therefore, it is important to determine the causes of its reduction in streamflow supply. Based on an analysis of changes in hydro-meteorological regime and the effects of direct human activities on streamflow, their contributions to the decrease in streamflow in different periods (natural period, 1964–1988; transition period, 1989–2002; post-transition period, 2003–2012) from the Hotan River to the Tarim River were quantified based on a double-mass curve of inflow and outflow in the Hotan River Basin. The results indicate that climatic changes reduced streamflow during the transition period yet increased streamflow during the post-transition period. Human activities reduced output by 73% in the transition period and by 127% in the post-transition period, which was significantly stronger than the contribution from the climate. Thus, human activities appear to be the main driving factor for the drop in streamflow from the Hotan River into the Tarim River. These results can be used as a reference for water resource planning and management.