•Three new steroidal glycosides were isolated from Marsdenia tenacissima.•All compounds were prepared and evaluated inhibitory activity against nitric oxide in RAW276.4 macrophages.•They exhibited weak inhibitory effects against nitric oxide (NO) at concentration 1 μM.Three new compounds 1–3 as C21 steroidal glycosides of diester derivatives of tenacigenin B were isolated from the ethanolic extract of stems of Marsdenia tenacissima. All compounds were prepared and evaluated inhibitory activity of nitric oxide in RAW276.4 macrophages. Compounds 1–3 exhibited inhibitory effects against nitric oxide (NO).

•Three new steroidal glycosides were isolated from Marsdenia tenacissima.•All compounds were prepared and evaluated inhibitory activity against nitric oxide in RAW276.4 macrophages.•They exhibited weak inhibitory effects against nitric oxide (NO) at concentration 1 μM.Three new compounds 1–3 as C21 steroidal glycosides of diester derivatives of tenacigenin B were isolated from the ethanolic extract of stems of Marsdenia tenacissima. All compounds were prepared and evaluated inhibitory activity of nitric oxide in RAW276.4 macrophages. Compounds 1–3 exhibited inhibitory effects against nitric oxide (NO).