This study was conducted to determine the optimal conditions of growth medium, temperature, and light quality for efficient propagation of Lygodium japonicum spores. The rate of spore germination and prothalium development was high in Knop and 1/8MS and 1/4MS media, which had low mineral content; in particular, the germination rate exceeded 74%, and the germinated spores developed into heart-shaped prothallia. However, in Knop‘s medium with the lowest mineral content, a rapid prothallium senescence was observed; in 1/4MS medium, prothallium development was delayed. Germination rate increased with the increase in temperature and reached its maximum, 86.7%, at 30°C; however, at this temperature, the prothallia were thinner and abnormal development of rhizoids was observed compared to normally developed prothallia and rhizoids at 25°C. Therefore, the results suggested that the optimal temperature for L. japonicum spore germination was 25°C. The rate of germination was also measured under different light conditions, and the highest rate of 90.6% was observed under LED red light compared to fluorescent (77.2%) or LED blue (5.4%) lights. The germinated spores developed into heart-shaped prothallia under LED red light; however, 15 days after seeding, prothallium development decreased and the became elongated. In contrast, a normal and continuous development of heart-shaped prothallia was observed under fluorescent light.