Plantainoside D (PD) is a potential anti-hypertensive active ingredient newly isolated from the dried plants of Chirita longgangensis var. hongyao. A sensitive and specific LC-ESI-MS/MS method was first developed and validated for the analysis of PD in rat plasma using genistein as the internal standard (IS). The plasma samples were pretreated with methanol-acetonitrile (50:50, v/v) to precipitate protein, and then chromatographed on a reverse-phase Agilent Zorbax XDB C18 column (50 mm × 2.1 mm, 3.5 μm). Gradient elution was utilized, with a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid, and the flow rate was set at 0.50 mL/min. The analytes were monitored by tandem-mass spectrometry with negative electrospray ionization. The precursor/product transitions (m/z) in the negative ion mode were 639.2 → 160.9 Thomson (Th) and 268.9 → 158.9 Thomson (Th) for PD and IS, respectively. Linearity was achieved in the 0.10-200 ng/mL range, with a lower limit of quantification of 0.10 ng/mL. The precision and accuracy for both intra- and inter-day determination of the analyte were all within ±15%. The present method has been applied for pharmacokinetic study of PD after oral and intravenous administration in rats. The oral absolute bioavailability (F) of PD in rats was estimated to be 1.12% ± 0.46% with an elimination half-life (t1/2) value of 1.63 ± 0.19 h, suggesting its poor absorption and/or strong metabolism in vivo.