Skeletal muscle contractility and myosin function decline following ovariectomy in mature, female mice. In the present study we tested the hypothesis that estradiol-replacement can reverse those declines. Four month-old female C57BL/6 mice (n=69) were ovariectomized (OVX) or sham-operated (Sham). Some mice were treated immediately with placebo or 17-estradiol (OVX+E₂) while other mice were treated 30 days post-surgery. Thirty or sixty days post-surgery, soleus muscles were assessed in vitro for contractile function and susceptibility to eccentric contraction-induced injury. Myosin structural dynamics was analyzed in extensor digitorum longus (EDL)muscles by electron paramagnetic resonance spectroscopy. Maximal isometric tetanic force was affected by estradiol status (P<0.001) being ~10% less in soleus muscles from OVX compared with Sham mice [168 mN (SD 16.7) vs. 180 mN (SD 14.4)] and was restored in OVX+E₂ mice [187 mN (SD 17.6)]. The fraction of strong-binding myosin during contraction was also affected (P=0.045) and was ~15% lower in EDL muscles from OVX compared with OVX+E₂ mice [0.263 (SD 0.034) vs. 0.311 (SD 0.022)]. Plasma estradiol levels were correlated with P_o (r=0.458; P<0.001) and active stiffness (r=0.329; P=0.044) indicating that circulating estradiol influenced muscle and myosin function. Estradiol was not effective in protecting muscle against an acute eccentric contraction-induced injury (P0.401) but did restore ovariectomy-induced increases in muscle wet mass caused by fluid accumulation. Collectively, estradiol had a beneficial effect on female mouse skeletal muscle.