Rapid population growth and urban expansion in the Yellow River Basin have aggravated land use transition and landscape fragmentation, resulting in a series of severe problems, such as the degradation of ecosystem function, reduction of biodiversity function, and the fragmentation of habitat. Exploring the spatio-temporal characteristics and causes of landscape fragmentation in the Yellow River Basin has important guiding significance for maintaining the stability of ecosystem, ecological restoration, and ecological civilization construction in the Yellow River Basin. However, previous studies lacked the knowledge of spatiotemporal characteristics and formation mechanism of landscape fragmentation, which is difficult to provide scientific guidance for the ecological governance and landscape pattern optimization of the Yellow River Basin. Based on Fragstats4.2 software, this study analyzed the spatiotemporal pattern of landscape fragmentation at county level of the Yellow River Basin from 2000 to 2018, and explored the formation mechanism of spatiotemporal differentiation of landscape fragmentation with the help of geodetector model. The results showed that:(1) patch density(PD) of the Yellow River Basin had no significant change during the study period. The edge density (ED), landscape shape index (LSI), and patch area mean (Area_AM) showed a trend of increasing at first and then decreasing. The aggregation index (AI) showed a trend of decreasing at first and then increasing. The index of contagion (CONTAG) continued to decrease, while the landscape division index (DIVISION) and shannon's diversity index (SHDI) increased gradually. (2) The degree of landscape fragmentation in the Yellow River Basin increased gradually during the study period. The degree of landscape fragmentation in the middle and lower reaches of the Yellow River Basin changed sharply, while the degree of landscape fragmentation in the upper reaches of the Yellow River Basin tended to be stable. (3) According to the results of geodetector, the fragmentation of the landscape in the Yellow River Basin was affected by multiple factors such as nature and society, among which there was a significant interaction enhancement effect. The main influencing factors of different regions are obviously different, while the intensity of human activities is the leading one. The results can provide ideas for exploring the formation mechanism of landscape fragmentation in the river basin, and have important referential significance to the ecological governance, ecosystem protection, and land use planning formulation of the Yellow River Basin.