Pregnenolone is a precursor/metabolic intermediate in the biosynthesis of steroid hormones. In this study, two kinds of pregnenolone compounds, i.e. 3β-acetoxypregna-5-en-20-one and 3β-acetoxypregna-5-en-20-hydroxy, were synthesized by solid-state NMR spectroscopy to investigate its chelation with Mg2+. To demonstrate, solid-state {1H}/13C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy was applied to a series of pregnenolone molecules that were incubated with Mg+2 under different concentrations. The high-resolution 13C NMR spectra of the steroid/Mg+2 mixtures exhibit two distinct 13C spectral patterns; one is attributed to pregnenolone in the free from Mg+2, and the other is due to the Mg+2 in the bound form. Based on the chemical shift analyses, we claimed that the Mg+2 might have associated with the oxygen attached to C20 via lone-pair electron, which induced a conformation change in pregnenolone. In this solid-state NMR study, we demonstrated that both 3β-acetoxypregna-5-en-20-one and 3β-acetoxypregna-5-en-20-hydroxy might adopt different steroidal ring conformations upon chelation with Mg+2.