Cibotium barometz (L.) J. Sm., an important taxa of the rare ornamental ferns, is listed in the Category II of State Key Protected Wild Plants. In present study, spores were used as materials to investigate the effects of different environmental factors (cultivation density, exogenous gibberellin, light quality) on the sex differentiation of C. barometz gametophytes, which could provide technical support for artificial propagation of C. barometz and study on sex determination mechanism of fern gametophytes. The results showed that: (1) The low density of cultivation (1 individual /cm², 5 individuals /cm²) is beneficial for the formation of archegonia and female gametophytes. With cultivation density increasing, number of archegonia per gametophyte and rate of female gametophyte decreased, in the meantime, number of antheridia per gametophyte and rate of male and hermaphroditic gametophyte increased, but excessive cultivation density (80 individuals /cm²) resulted in a mass of asexual gametophytes. (2) With the cultivation time prolonging, the rate of hermaphroditic gametophyte increased in the different cultivation density. (3) Exogenous gibberellin A₄ (GA₄) significantly suppressed the formation of archegonia and female gametophytes, and promoted the formation of antheridia and male gametophytes. However, exogenous gibberellin A₃ (GA₃) had no significant effect on the sex differentiation of C. barometz gametophytes. (4) Light quality (white, red, blue) had no significant effect on the sex differentiation of C. barometz gametophytes, but had some effect on gametophyte development and morphogenesis.