Recently, there has been increasing evidence showing that males estimate whole numbers more accurately than females on the number line. However, relatively little is known about what factors contribute to this gender gap. The current study explored potential mediators of the gender difference in number line estimation, including spatial skills and spatial anxiety. In the Fall (time-point 1 [T1]), 490 children from kindergarten through fourth grade (274 girls) completed age-appropriate measures of number line estimation, spatial skills (including proportional reasoning, mental rotation, mental transformation, and visuospatial working memory), and spatial anxiety. About 5 month later in the Spring (time-point 2 [T2]), children completed the same measure of number line estimation again. Boys were more accurate on number line estimation, proportional reasoning, and mental rotation than girls, whereas girls showed higher levels of spatial anxiety. Critically, spatial skills (a latent variable constructed from proportional reasoning, mental rotation, mental transformation, and visuospatial working memory) at T1 mediated the gender difference in T2 number line estimation whereas spatial anxiety was not a significant mediator. These relationships held even after controlling for T1 number line estimation, reading achievement, and reading anxiety. Among the four spatial skills, proportional reasoning and mental rotation (but not mental transformation or visuospatial working memory) were mediators of the gender difference in T2 number line estimation. These findings constitute, to our knowledge, the first evidence regarding factors contributing to the gender difference in whole number line estimation. (PsycInfo Database Record (c) 2022 APA, all rights reserved).