Abstract: To determine the influence of different culture mediums and spore dimensions on the sex differentiation of Ceratopteris thalictroides,we used MS solid medium,modified knop’s solid medium,tap water,and sterile distilled water to culture median sized spores and modified knop’s solid medium to culture three different sized spores.The germination and sex differentiation were observed and recorded.Results showed that second rank spores (equatorial axis 120140 μm) had the highest germination rate. First rank spores (equatorial axis>140 μm) were inclined to form hermaphroditic gametophytes and the third rank spores (equatorial axis<120 μm) were inclined to form the male gametophytes.Spore germination was better in MS solid medium and modified knop’s solid medium than in tap water and sterile distilled water.The proportion of hermaphroditic gametophytes in different mediums,from the highest to the lowest,was MS solid medium,modified knop’s solid medium,tap water,and sterile distilled water,while the proportion of male gametophytes showed the opposite pattern.This experiment provides theory data on the introduction,preservation,artificial breeding,and molecular biology of Ceratopteris thalictroides.