The impacts of climate variability and anthropogenic activities on hydrological processes have been of wide concern in the hydrology community during recent decades. In this study, specific investigations of individual impacts of climate variability and anthropogenic activities on runoff during 1964–2010 are conducted for the upper Huaihe River Basin at Huaibin (HB) and its five sub-catchments. The non-stationary relationship between precipitation and runoff was firstly analyzed, and according to change point detection results, long-term series for each catchment was divided into pre-change period and post-change period, respectively. Then, the climate variability and human activities that occurred in the whole HB catchment were analyzed. Finally, using two quantitative evaluation methods, the individual impacts of climate variability and human activities for each catchment were assessed. The results showed that for the whole HB catchment, runoff changes during the whole post-change period are mainly attributed to climate variability, as for its sub-catchments except the Xinxian catchment. As for decadal behaviors, runoff generally suffered more human-induced impacts in dry decades (1990s) than wet decades (1980s and 2000s). These results reflected the complex role of climate variability and human activities in influencing the runoff regime, which could be considered in local water resources management.