Aim The purpose of this study was to determine whether 17β‐Estradiol (E2) enhances the activation, proliferation and differentiation of muscle satellite cells (SC) following eccentric exercise either via Insulin‐like Growth Factor‐1 (IGF‐1) or through phosphatidylinositol 3‐kinase (PI3K) signaling. Methods This study used 64, nine‐week old, ovariectomized Sprague‐Dawley rats that were divided into eight treatments groups based on: estrogen status (0.25 mg estrogen pellet or sham), exercise status (90 min run @ 17 m/min, ‐13.5° or unexercised), PI3K signaling inhibition (0.7 mg Wortmannin·kg−1 Body Weight or DMSO control). Results Significant increases in total SCs were found in both soleus and white gastrocnemius muscles (immunofluorescent co‐localization of Pax7+ nuclei) 72 hr following eccentric exercise (p < 0.05). Estrogen‐supplementation caused a further enhancement in total SCs in exercised rats (p < 0.05). In animals where the PI3K pathway was inhibited, regardless of estrogen or exercise status, there was no significant enhancement of SC number in both the soleus or white gastrocnemius muscles. Interestingly, estrogen‐supplementation lowered muscle levels of IGF‐1 with this effect being most prominent in the soleus muscle. While IGF‐1 was increased following exercise (p < 0.05), estrogen‐supplementation abrogated this increase back to sedentary levels. Conclusion This data suggests that the increase in SC population following exercise in estrogen‐supplemented females may be mediated via PI3K pathway signaling and not IGF‐1. This article is protected by copyright. All rights reserved.