According to the consistency analysis of meteorological and hydrological series, one natural period (1961-1979) and three impact periods (① 1980-1989, ② 1990-1999 and ③ 2000-2016) have been divided by the Mann-Kendall's test in the Yixun River Basin. The variable infiltration capacity (VIC) model calibrated by the streamflow data in natural period and stepwise method was used to identify the fluctuant impacts of climate change and human activities on the river runoff. Results showed that the climate change in the study area at annual scale was mainly reflected in the significant increase in temperature and there was no obvious trend in precipitation, while the runoff indicated a notable descending trend. Furthermore, the significant increase of precipitation in non-flood season was found at seasonal scale. Climate change and human activities would have significant effects on runoff with the complex mechanism; stepwise method was more appropriate for analyzing the fluctuant impacts than traditional methods. In the period ② with abundant precipitation, the increase of winter rainfall would significantly increase runoff. However, in the period with less precipitation, such as periods ① and ③, the increase of evaporation and the decrease of soil water content would be two factors causing the reduction in runoff. Water consumption reduced runoff in periods ① and ③, and the impact of human activities gradually increased. In period ②, urbanization would be the major effect of the ascending on the runoff. The studies on how climate change and human activities affect the runoff could provide theoretical principal for water resources management and planning in future.