Abstract: Using separation methods such as silica gel, MCI, ODS, Sephadex LH-20 gel column chromatography, and high-performance liquid chromatography, two C21 steroid components were isolated and identified from the traditional medicinal plant Munronia unifoliolata Oliv. (commonly known as "Aituotuo"), which is used for treating rheumatic joint pain and traumatic injuries. The structures of these compounds were identified as 2α, 3α, 15β-trihydroxy-20(S)-benzoyl-pregnane (1) and 2α, 3α, 15β-trihydroxy-20(S)-tigloyl-pregnane (2) using comprehensive structural identification methods, including UV, IR, high-resolution mass spectrometry, and one-dimensional and two-dimensional nuclear magnetic resonance. Compounds 1 and 2 were reported for the first time as pregnane-type components from M. unifoliolata, with compound 1 being a new compound. The results of the bioactivity screening indicated that both compound 1 and compound 2 did not inhibit nitric oxide (NO) production and showed no activity in reversing multidrug resistance.