To determine whether $17{eta}$-estradiol regulates peroxisome proliferator-activated receptor ${alpha}$ ($PPAR{alpha}$)-mediated lipid reduction in skeletal muscle cells and tissues and to investigate the molecular mechanism involved in this process, C2C12 myotubes and female ovariectomized C57BL/6J mice were treated with combinations of the $PPAR{alpha}$ agonist fenofibrate or $17{eta}$-estradiol. Both differentiated C2C12 myotubes and skeletal muscle of high-fat diet-fed obese mice showed lipid accumulation. However, either fenofibrate or $17{eta}$-estradiol decreased lipid droplets and increased mRNA levels of $PPAR{alpha}$ target enzymes responsible for fatty acid oxidation. When mice and myotubes were concomitantly treated with fenofibrate and $17{eta}$-estradiol, $17{eta}$-estradiol reversed the effects of fenofibrate on lipid accumulation and mRNA expression of $PPAR{alpha}$ target genes. In addition, $17{eta}$-estradiol not only decreased basal levels of $PPAR{alpha}$ reporter gene activation but also reduced fenofibrate-induced luciferase reporter activity in C2C12 myoblasts. Moreover, $17{eta}$-estradiol inhibited the recruitment of the $PPAR{alpha}$ coactivator CREB-binding protein. These results demonstrate that $17{eta}$-estradiol inhibits the actions of $PPAR{alpha}$ on skeletal muscle lipid metabolism by down-regulating the expression of $PPAR{alpha}$ target genes for fatty acid catabolism, which may be mediated through the inhibition of $PPAR{alpha}$-dependent coactivator recruitment.