Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their anti-oxidant activity. Here, we investigated the anti-inflammatory effects and underlying mechanisms of Asp and Not against lipopolysaccharide (LPS)-mediated vascular inflammatory responses. The anti-inflammatory activities of Asp and Not were determined by measuring permeability, monocytes adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells (HUVECs) and mice. We found that each compound inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of neutrophils to human endothelial cells. Asp and Not also suppressed LPS-induced hyperpermeability and leukocyte migration in vivo. Furthermore, each compound suppressed the production of tumor necrosis factor-α (TNF-α) or interleukin (IL)-6 and the activation of nuclear factor-κB (NF-κB) or extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, treatment with each compound resulted in reduced LPS-induced lethal endotoxemia. These results suggest that Asp and Not posses anti-inflammatory functions by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.