AimsThe aim of this study was to characterize a fungal endophyte Y3 from pigeon pea (Cajanus cajan [L.] Millsp), as a novel producer of vitexin, and its culture medium optimization and antioxidant activity. Methods and ResultsThe endophyte from the leaves of pigeon pea was identified as Dichotomopilus funicola by the morphological and molecular characteristics. The most important medium variables affecting vitexin production in liquid culture of D. funicola Y3 were screened by Plackett-Burman design, and three culture medium constituents (i.e. l-phenylalanine, salicylic acid and CuSO(4)5H(2)O) were identified to play significant roles in vitexin production. The most significant factors were further optimized using by central composite design with response surface methodology. The DPPH radical-scavenging assay indicated that fungal vitexin exhibited notable antioxidant activity with an EC50 value of 164gl(-1). ConclusionsFirst, a novel endophyte vitexin-producing Dichotomopilus funicola Y3 was isolated from pigeon pea (Cajanus cajan[L.] Millsp.). The maximum vitexin yield was obtained as7886mgl(-1) under the optimum culture medium constituents: 006gl(-1)l-phenylalanine, 021gl(-1) salicylic acid, and 019gl(-1) CuSO(4)5H(2)O in medium, which is 459-fold higher than that in the unoptimized medium. Also, fungal vitexin clearly demonstrated its antioxidant potential. Significance and Impact of the StudyThese findings provide an alternative source for large-scale production of vitexin by endophytic fungal fermentation and have a promising prospect in food and pharmaceutical industry.