Andrographolide is widely used in clinic as an anti-inflammatory and antibiotic drug. In this paper, the metabolites of andrographolide in rats after single oral doses of 120 mg/kg were investigated. The structures of the metabolites were elucidated by high-resolution mass spectra, NMR spectroscopy including 1H NMR, 13C NMR, and two-dimensional NMR, through comparison to a synthetic standard. The main metabolite of andrographolide in rats was 14-deoxy-12(R)-sulfo andrographolide. In the proposed mechanism, the beta-carbon of alpha, beta-unsaturated carbonyl was attacked by sulfonic acid, to form the sulfonate compound. This was a rare metabolic reaction. It may be the main metabolic pathway of andrographolide in rats. The polarity of the sulfonate metabolite increased greatly and could be easily eliminated from body.