The water shortage in the Huaihe River Basin (HRB), China, has been aggravated by population growth and climate change. To identify the characteristics of streamflow change and assess the impact of climate variability and human activities on hydrological processes, approximately 50 years of natural and observed streamflow data from 20 hydrological stations were examined. The Mann–Kendall test was employed to detect trends. The results showed the following. (i) Both the natural and the observed streamflow in the HRB present downward trends, and the decreasing rate of observed streamflow is generally faster than that of the natural streamflow. (ii) For the whole period, negative trends dominate in the four seasons in the basin. The highest decreasing trends for two kinds of streamflow both occurred in spring, and the lowest ones were in autumn and winter. (iii) Based on the above analysis and quantifying assessment for streamflow decrease, human activity was the main driving factor in the Xuanwu (80.78%), Zhuangqiao (79.92%), Yongcheng (74.80%), and Mengcheng (64.73%) stations which all belong to the Huaihe River System (HRS). On the other hand, climate variability was the major driving factor in the Daguanzhuang (68.89%) and Linyi (63.38%) stations which all belong to the Yishusi River System (YSR).