Anthropogenic habitat modification and fragmentation is considered one of the most serious threats to biodiversity. To develop effective conservation strategies towards such pressures we need to improve our understanding of how species richness and community composition are shaped by species’ responses to landscape patterns. In this study we tested relationships between patch size and isolation, species richness and species traits in a fragmented landscape of calcareous grasslands—a diversity hotspot in Southern Norway. We recorded a total of 381 vascular plant species, of which 50 are considered habitat specialists, distributed among 86 habitat patches (50–9,475 m2) in 22 of 50 randomly sampled 500 × 500 m-plots. We found that large habitat patches held more species of both generalists and specialists than small habitat patches and that well-connected patches held more specialist species than isolated patches. About 1/3 of the habitat specialists in this study system showed vulnerability to isolation, i.e. lower probability of occurrence in isolated patches. Traits related both to persistence (short lifespan) and colonization ability (low seed production per plant) were predictors of vulnerability to isolation. Our results indicate that both colonization and extinction processes affect species composition and richness, and that the rescue-effect—mitigation of local, area-dependent extinctions through colonization—is reduced in isolated patches. These findings suggest that conservation strategies should place greater emphasis on the spatial configuration of the habitat network, and on the preservation of colonization processes to ensure regional persistence of species.