Moderate physical training has been shown to hinder age-related memory decline. While the benefits of physical training on hippocampal memory function are well-documented, little is known about its impact on working memory, which is linked to the prelimbic cortex (PrL), one major subdivision of the prefrontal cortex. Here, we examined the effects of physical training on spatial working memory in a well-established animal model of physical training, starting at 16 months of age and continuing for 5 months (running wheel 1 h/day and 5 days/week). This training strategy improved spatial working memory in aged mice (22-month-old), which was accompanied by an increased spine density and a lower TAF15 expression in the PrL. Specifically, physical training affected both thin and mushroom-type spines on PrL pyramidal cells, and prevented age-related loss of spines on selective segments of apical dendritic branches. Correlation analysis revealed that increased TAF15-expression was detrimental to the dendritic spines. However, physical training downregulated TAF15 expression in the PrL, preserving the dendritic spines on PrL pyramidal cells and improving working memory in trained aged mice. When TAF15 was overexpressed in the PrL via a viral approach, the benefits of physical training on the dendritic spines and working memory were abolished. These data suggest that physical training at a moderate pace might downregulate TAF15 expression in the PrL, which favors the dendritic spines on PrL pyramidal cells, thereby improving spatial working memory.