Activation of microglia induces the production of various inflammatory mediators including nitric oxide (NO), leading to neurodegeneration in many central nervous system diseases. In this study, we examined the effects of chalcone glycosides isolated from Brassica rapa L. 'hidabeni' on lipopolysaccharide (LPS)-induced NO production using rat immortalized microglia HAPI cells. 4'-O-β-D-Glucopyranosyl-3',4-dimethoxychalcone (A2) inhibited LPS-induced inducible NO synthase (iNOS) expression and NO production. However, A2 did not affect nuclear factor-κB and mitogen-activated protein kinase pathways. The signal transduction and activator of transcription 1 (STAT1), which is activated via production of IFN-β by LPS, is an important transcription factor responsible for LPS-induced iNOS expression. A2 suppressed LPS-induced phosphorylation and nuclear translocation of STAT1, although it had no effects on LPS-induced IFN-β expression. These results indicate that the inhibitory effect of A2 is due to the prevention of STAT signaling. Moreover, structure-activity relationship studies on newly synthesized 'hidabeni' chalcone derivatives showed that 4'-O-β-D-glucopyranosyl-3'-methoxychalcone (A11), which has no functional groups in the B-ring, inhibits LPS-induced NO production more potently than A2.