The vertical stratification of the stand may lead to a high heterogeneity of microenvironment in the forest, which further influences the understory regeneration and succession of the forest. Most relevant previous studies emphasized the overall effects of the Whole-stand structural characteristics on understory regeneration, while the strata-specific impacts of the overstory should be explored especially for those forests with a complicated combination of overstory species and heights. In this study, a subtropical evergreen broad-leaved forest in Tianmu Mountain of China was intensively investigated within 25 plots of 20 m × 20 m, aiming to find out how significant the stratified overstory (trees with diameter at breast height (DBH) ≥ 5 cm) structure and non-structure characteristics impact the understory (trees with DBH < 5 cm) regeneration. Regardless of species composition, the studied overstory was evenly divided into three strata (i.e. upper, middle and lower strata) according to their heights. Redundancy analysis was applied to explore both overall and strata-specific forest structure on characteristics (height, DBH, species diversity, and density) of tree regeneration. We found that the overall effect of the whole overstory on the forest regeneration depended mostly on diameter at breast height (DBH), tree species richness index and crown width. However, when analyzing with the strata-specific characteristics, the most pronounced impact factors for the regeneration were tree height of the upper and lower forest strata, tree species richness index and crown width of the middle and lower forest strata, and the competition index impact of the lower forest stratum. Among the three strata, the lower forest stratum showed the most significant impact with three characteristics on the understory regeneration, which may be attributed to their direct competition within the overlapping near-ground niches. Among the new generations, seedlings and saplings were more sensitive to the overstory structural characteristics than young trees. Our results suggest that the overstory showed strata-specific effects on the understory regeneration of evergreen broad-leaved forests in subtropical China, which provides theoretical basis for strata-specific forest management in similar forests.