[Objective] The variation of the observed runoff and natural runoff of the Yellow River was analyzed to explore the impact of human activities on runoff in the study area.[Methods] Based on the natural runoff and observed runoff of eight hydrological stations in the main stream of the Yellow River from 1969 to 2018, the variation of the observed natural runoff in the last 50 years was analyzed using a Mann-Kendall trend test and mutant test. In addition, the impact of human activities on runoff was considered based on the water consumption data of each region in the last 15 years.[Results] ① The observed runoff in the upper, middle, and lower reaches of the Yellow River all decreased from 1969 to 2018. The average annual reduction rates at the Lanzhou, Huayuankou, and Lijin stations were 51, 355, and 413 million m³/a, respectively. ② In the last 50 years, the greatest alteration of natural runoff and observed runoff occurred during 1986 and 1990. Combined with previous studies and important water events of the Yellow River, the runoff alteration in 1986 may have been related to the implementation of soil and water conservation measures since 1984 as well as the construction of Longyangxia Reservoir in 1986. The runoff alteration in 1990 may have been due to the surge of water consumption in the 1980s and 1990s in addition to the underlying surface water variation of the basin. ③ The difference between the natural and observed runoff gradually increased from the upper reach to the lower reach, which was generally consistent with the increased average water consumption over the past 15 years. In addition, the annual average observed runoff only accounted for 42% of natural runoff at Lijin Station. The two regions with the highest water consumption were the lower part of Huayuankou and the area between Lanzhou and Toudaoguai, reaching 1.06×10¹⁰ m³ and 1.04×10¹⁰ m³, respectively.[Conclusion] The main reason for the large difference between the natural and observed runoff in the area below Lanzhou station was the water consumption associated with human activities, especially for farmland irrigation. Therefore, it is necessary to promote the development of water-saving agriculture in the Yellow River basin and to reasonably allocate water consumption.