The three-dimensional (3D) spatial structure within cities can reveal more information about land development than the two-dimensional spatial structure. Studying the relationship between the urban 3D spatial structure and the population distribution is a crucial aspect of the relationship between people and land within cities. However, a few relevant studies focus on the differences between employment population and night population distribution in relation to urban 3D spatial structure. Therefore, this study proposes a new concept of 3D space-filling degree (3DSFD), which is applicable to evaluate the city’s 3D spatial structure. We took 439 blocks in Kunming’s Main Urban District as a sample and analyzed the 3D spatial structure based on geographic information data at the scale of a single building. The characteristics and differences of the daytime and night population distribution in Kunming’s Main Urban District were identified using cell phone signaling big data. Accordingly, a cross-sectional dataset of the relationship between the city’s 3D spatial structure and the population distribution was constructed, with the 3D space-filling degree of the block as the dependent variable, two indicators of population distribution (daytime and night population density) as the explanatory variables, and seven indicators of distance from the city center, and building, road, and functional place densities, proportion of undevelopable land area, housing prices, and land use type as the control variables. We used spatial regression models to explore the significance, strength, and direction of the relationship between urban 3D spatial structure and population distribution. We found that the spatial error model (SEM) was the most effective. The results show that only night population distribution is significantly and positively related to 3DSFD. Every 1% increase in night population density in a block will increase the value of 3DSFD by 2.8307%. The night population distribution is the core factor affecting the 3D spatial structure of Kunming’s Main Urban District. The correlation between daytime population distribution and 3DSFD is not significant. This variability has been ignored in previous studies. The findings are informative for further understanding of the relationship between urban 3D space and population distribution, especially the difference between night and daytime populations. This study can help city managers reasonably plan urban land development intensity and construction height, guide the population layout and formulate management policies to improve urban population and space matching, enhancing the livability and attractiveness of cities.
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