There is little understanding of successional dynamics of N fixation in northern boreal forests. Recent evidence suggests that N fixation by cyanobacteria in association with the common feather moss Pleurozium schreberi contributes to a significant proportion of the total N economy. The purpose of the work herein was to determine how time since last fire influences N fixation rates in boreal forests. We evaluated seasonal N fixation rates on a total of 12 natural forest preserves varying in time since last fire (35–355 years). Each site was monitored for N fixation activity using a calibrated acetylene reduction assay. Nitrogen fixation rates were found to increase linearly with time since fire. This increase in N fixation with succession is likely a function of degree of colonization by cyanobacteria and site factors such as presence of available N. Surface applications of 4.5 kg N·ha−1·yr−1 as NH4NO3 were found to eliminate N fixation while applications of P resulted in only a slight and temporary increase of N fixation rates. In contrast to common observation our findings suggest that N fixation in boreal forests becomes more important in late succession. Limited N availability in late succession is clearly one of the primary drivers of N fixation rates in boreal forest ecosystems. These findings may help to explain the origin of high rates of net N accumulation in soil unaccounted for at northern boreal sites.