Aims The aim was to isolate, identify and characterize endophytes from pigeon pea (Cajanus cajan [L.] Millsp.), as novel producer of cajanol and its in vitro cytotoxicity assay. Methods and Results Isolation, identification and characterization of novel endophytes producing cajanol from the roots of pigeon pea were investigated. The endophytes were identified as Hypocrea lixii by morphological and molecular methods. Cajanol produced by endophytes were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). R-18 produced the highest levels of cajanol (322 center dot 4 +/- 10 center dot 6gl-1 or 102 center dot 8 +/- 6 center dot 9gg-1 dry weight of mycelium) after incubation for 7days. The cytotoxicity towards human lung carcinoma cells (A549) of fungal cajanol was investigated in vitro. Conclusions First, a novel endophyte Hypocrea lixii, producing anticancer agent cajanol, was isolated from the host pigeon pea (Cajanus cajan [L.] Millsp.). Fungal cajanol possessed stronger cytotoxicity activity towards A549 cells in time- and dose-dependent manners. Significance and Impact of the Study This endophyte is a potential handle for scientific and commercial exploitation, and it could provide a promising alterative approach for large-scale production of cajanol to satisfy new anticancer drug development.