Display omitted ► Solid-state NMR provides a sensitive tool for analyzing steroid hormone conformation. ► 17β-Estradiol reveals singlet patterns in the 13C NMR spectra. ► 17α-Estradiol reveals multiplet patterns in the 13C NMR spectra. ► 17α- and 17β-estradiol are flexible to adopt different conformations in the lipid environment. Solid-state { 1H} 13C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to 17β-estradiol (E2) and 17α-estradiol (E2α), to analyze the steroidal ring conformations of the two isomers in the absence and presence of lipids at the atomic level. In the absence of lipid, the high-resolution 13C NMR signals of E2 in a powdered form show only singlet patterns, suggesting a single ring conformation. In contrast, the 13C signals of E2α reveal multiplet patterns with splittings of 20–300 Hz, implying multiple ring conformations. In the presence of a mimic of the lipid environment, made by mixing 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl- sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, E2 and E2α revealed multiplet patterns different from those seen in the absence of lipids, indicating that the two isomers adopt multiple conformations in the lipid environment. In this work, on the basis of chemical shift isotropy and anisotropy analysis, we demonstrated that E2 and E2α prefer to adopt multiple steroidal ring conformations in the presence of a lipid environment, distinct from that observed in solution phase and powdered form.