Aging is accompanied by inexorable loss of muscle tissue. One of the underlying causes for this is the massive change in the hormonal milieu of the body. The role of a female sex steroid – estrogen – in these processes is frequently neglected, although the rapid decline in its production coincides with a steep deterioration in muscle performance. We recruited 54- to 62-year-old monozygotic female twin pairs discordant for postmenopausal hormone replacement therapy (HRT, n = 11 pairs; HRT use 7.3 ± 3.7 years) from the Finnish Twin Cohort to investigate the association of long-term, estrogen-based HRT with skeletal muscle transcriptome. Pathway analysis of muscle transcript profiles revealed significant HRT-induced up-regulation of a biological process related to regulation of cell structure and down-regulation of processes concerning, for example, cell–matrix interactions, energy metabolism and utilization of nutrients (false discovery rate < 0.15). Lending clinical relevance to the findings, these processes explained a significant fraction of the differences observed in relative proportion of muscle within thigh and in muscle performance (R2 = 0.180–0.257, P = 0.001–0.023). Although energy metabolism was affected through down-regulation of the transcripts related to succinate dehydrogenase complex in mitochondria, no differences were observed in mtDNA copy number or oxidative capacity per muscle cross section. In conclusion, long-term use of HRT was associated with subtle, but significant, differences in muscle transcript profiles. The better muscle composition and performance among the HRT users appeared to be orchestrated by improved regulatory actions on cytoskeleton, preservation of muscle quality via regulation of intramuscular extracellular matrix and a switch from glucose-oriented metabolism to utilization of fatty acids.