Agricultural improvement of seminatural grasslands has been shown to result in changes to plant and microbial diversity, with consequences for ecosystem functioning. A microcosm approach was used to elucidate the effects of two key components of agricultural improvement (nitrogen addition and liming) on ammonia-oxidising bacterial (AOB) communities in an upland grassland soil. Plant species characteristic of unimproved and improved pastures (A. capillaris and L. perenne) were planted in microcosms, and lime, nitrogen (NH4NO3), or lime plus nitrogen added. The AOB community was profiled using terminal restriction fragment length polymorphism (TRFLP) of the amoA gene. AOB community structure was largely altered by NH4NO3 addition, rather than liming, although interactions between nitrogen addition and plant species were also evident. Results indicate that nitrogen addition drives shifts in the structure of key microbial communities in upland grassland soils, and that plant species may play a significant role in determining AOB community structure.