Boreal peatlands are substantial sources of isoprene, a reactive hydrocarbon. However, it is not known how much mosses, vascular plants and peat each contribute to isoprene emission from peatlands. Furthermore, there is no information on the effects of declining water table depth on isoprene emission in these naturally wet ecosystems, although water table is predicted to decline due to climate warming. We studied the relative contribution of mosses vs. vascular plants to isoprene emission in boreal peatland microcosms in growth chambers by removing either vascular vegetation or both vascular vegetation and mosses. The microcosms represented wet hollows and dry hummocks of a boreal ombrotrophic bog. A water table drawdown treatment was applied to the hollows with naturally high water table. The mean (±SE) isoprene emission from hummocks with intact vegetation, 30 ± 6 μg m−2 h−1, was decreased by over 90% with removal of vascular plants or all vegetation. Thus, our results indicate that vascular plants, in contrast to mosses, were the main source of isoprene in the studied peatland ecosystem. Water table drawdown also significantly decreased the emissions; the mean isoprene emission from hollows with intact vegetation, 45 ± 6 μg m−2 h−1, was decreased by 25% under water table drawdown. However, water table drawdown reduced net ecosystem carbon dioxide (CO2) exchange more dramatically than isoprene emission. Isoprene emission strongly correlated with both CO2 exchange and methane emission. In conclusion, isoprene emissions from peatlands will decrease, but the proportion of assimilated carbon lost as isoprene will increase, if the naturally high water table declines under the changing climate.