A loop-mediated isothermal amplification (LAMP) assay was developed to detect Enterocytozoon bieneusi DNA for the first time from human faecal specimens. Four primers specific for Enterocytozoon bieneusi were designed corresponding to small subunit rRNA gene sequences and tested on 100 human faecal specimens. Thirty-nine of the faecal specimens (39 %) were confirmed positive for Enterocytozoon bieneusi by LAMP compared with 33 % by PCR and 32 % by light microscopy. LAMP yielded 94 % sensitivity and 88 % specificity compared with microscopy (sensitivity 48 %, specificity 76 %). No significant differences in positive detection of Enterocytozoon bieneusi were found among the three methods (P.0.05). However, LAMP has shown a substantial agreement with PCR (k50.78) and fair agreement was demonstrated between microscopy and PCR (k50.25). In conclusion, the LAMP assay proved to be useful as a simplified, rapid, sensitive and specific alternative molecular screening tool in the diagnosis of Enterocytozoon bieneusi in faecal specimens.