Astragalus chrysochlorus Boiss. & Kotschy (2n = 16) is one of the rare Turkish endemic species and it is listed in the Red Data Book of Turkish Plants as endangered. This species has been used traditionally for its wound healing properties and a crude ethanol extract prepared from the roots exhibits antioxidant and cytotoxic activities. In this study, a detailed phytochemical analysis was performed on A. chrysochlorus calli that resulted in the isolation of a major constituent. The purified molecule's structure elucidation was completed by spectral methods [nuclear magnetic resonance (NMR) and mass spectrometry (MS)], which revealed a rarely encountered the flavonoid in the Astragalus genus, nicotiflorin. In order to increasing nicotiflorin content in the callus cultures, the effects of culturing time and elicitor treatment were investigated. The HPLC analyses showed that the maximal production of nicotiflorin occurred with long-term cultured (13 years old) callus as 4775 mu g/g dry weight (DW), whereas it was 132 mu g/g DW for short-term cultured (2 months old) ones. Then, the 24 h treatment of the yeast extract that was used as biotic elicitor had negative effect on the production of nicotiflorin. The data obtained from this study could be significant for the mass production of nicotiflorin from long term in vitro cultured A. chrysochlorus callus.