Aim To investigate the bioactive metabolite of endophytic fungus from Kalimeris indica. Methods and Results A strain ZJLQ336 was separated from the leaves of K. indica. It was identified as Cadophora orchidicola based on the phylogenetic analysis of ITS-rDNA sequences. From the fermentation broth a metabolite cercosporamide (compound 1) was isolated, and its structure was determined by spectroscopic analysis. Additionally, this compound was subjected to bioactivity assays, including antifungal activity against seven plant pathogenic fungi, as well as its potential immunoregulatory effects on HEK-BLUE (TM)-hTLR4 cells, splenocytes and macrophages. The results showed that cercosporamide had strong growth inhibition against five common plant pathogenic fungi, including Pestalotia diospyri, Botrytis cinerea, Fusarium oxysporum, Sclerotium rolfsii and Penicillum digitatum with EC50 values of 5 center dot 29 x 10(-3), 0 center dot 61, 0 center dot 93, 2 center dot 89 and 6 center dot 7 mu g ml(-1) respectively. Among which S. rolfsii was one of the main pathogens in K. indica. Moreover, cercosporamide not only significantly stimulated TLR4 activation, splenocyte proliferation and production of cytokines, IFN-gamma and TNF-alpha, but also up-regulated the production of TNF-alpha and NO in RAW264.7 macrophages clearly. Conclusions This is the first report of endophytic C. orchidicola from K. indica and its metabolite cercosporamide. The results of pharmacological tests highlight the potential fungicide and TLR4 agonist of cercosporamide. Significance and Impact of the Study This study indicates endophytic fungi are good resources for natural bioactive metabolites. It also suggests that cercosporamide is a potential fungicide and TLR4 agonist.