Premise The genus Arisaema (Araceae) has rapidly diversified in Japan, and multiple species often coexist in the field. Although Japanese Arisaema species hybridize from artificial crossing, hybrid individuals are rare in mixed populations; suggesting the presence of effective pre-pollination barriers. We examined the following reproductive barriers between A. sikokianum and A. tosaense: habitat, phenology, and pollinator isolations. Methods Habitat isolation was examined by interspecific comparisons of microhabitat conditions at a mixed site and of altitude at the sampling site of herbarium specimens. Phenological isolation was evaluated by comparing seasonal transition in apparent spathe condition and frequency of insect visitation. Pollinator isolation was examined by comparing floral visitor assemblages between the two Arisaema species. To avoid overestimation of pollinator isolation due to seasonal changes in insect assemblages, we also compared visitor assemblages between natural and late-flowering A. sikokianum, where the latter was experimentally introduced and blooming with a natural A. tosaense population. Results Microhabitat conditions and sampling elevations of herbarium specimens overlapped between the two Arisaema species. At the population level, A. sikokianum and A. tosaense flowered for 39 and 52 days, respectively, with 13 days overlap. Insect visitation in A. sikokianum decreased before the seasonal overlap. Floral visitor assemblages differed between the two Arisaema species, while the difference between natural and late-flowering A. sikokianum was less distinct. Conclusions Phenological and pollinator isolation contribute to reproductive isolation between the two Arisaema species and should enable the two species to coexist in this area.