Huperzine A (HupA), a naturally occurring alkaloid in the plant family Huperziaceae, has drawn great interest for its potential application in Alzheimer disease therapy. Our primary objective was to identify alkaloid- and HupA-producing fungi from the Chinese folk herb, Huperzia serrata. We established a rapid and efficient model for screening HupA-producing endophytic fungal strains. The presence of HupA in Paecilomyces tenuis YS-13 was analysed by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. The fermentation yield of HupA was 21.0 mu g/L, and the IC50 of the crude extract of YS-13 fermentation broth was 1.27 +/- 0.04mg/mL. This is the first report of P. tenuis as a HupA-producing endophyte isolated from Huperziaceae.