AbstractFlowers play a central role in plant reproduction by dispersing and receiving pollen grains delivered by animal vectors or air. They are rich in various nutrients, and therefore, provide an ideal habitat for many microbes. Recent studies have revealed that flower microbial communities can be highly variable among species, individuals, or even floral parts. However, sexual differences in flower microbial communities have rarely been investigated. In this study, we analyzed the flower prokaryotic communities of a dioecious plant (male and female flowers are produced on different individuals), Mallotus japonicus, in its natural habitat in Otsu, Japan. Using 16S rRNA amplicon sequencing, we found a differentiation in microbial communities between male and female flowers. Procaryotes on male flowers were relatively diverse, and included several dominant amplicon sequence variants (ASVs), mainly from the Gammaproteobacteria. These ASVs were also found on the body surface of flower visitor insects, suggesting that the visitors dispersed these microbes when they visited the flowers. On the other hand, female flower samples were overwhelmingly dominated by a single bacterial sequence from the Alphaproteobacteria, which showed a peak of relative abundance at the middle of the flowering season. The bacterium had already been present at anthesis, and its relative abundance on flower visitors was low. Flower visitors may have little effect on the microbial composition on female flowers. The lower diversity of microbes on female flowers than on male flowers suggests that the female flowers function as a stronger filter, possibly related to a strong defense against antagonists. These differences may be associated with different reproductive strategies of male and female flowers. This study indicates that dioecious plants provide unique opportunities to study roles of microbes in the evolution of floral traits that have mostly been overlooked in pollination ecology.