Biochar has been frequently suggested as an amendment to improve soil quality and mitigate climate change. To investigate the optimal management of nitrogen (N) fertilization, we examined the combined effect of biochar and N fertilizer on plant N uptake and N2O emissions in a cereal rotation system in a randomized two-factorial field experiment on a sandy loam soil in Brandenburg, Germany. The biochar treatment received 10Mgha-1 wood-derived biochar in September 2012. Four levels of N fertilizer, corresponding to 0, 50%, 100%, 130% of the recommended fertilizer level, were applied in winter wheat (Triticum aestivum L.)) and winter rye (Secale cereal L.) in 2013 and 2014 followed by the catch crop oil radish (Raphanus sativus L. var. oleiformis). Biomass and N uptake of winter wheat and winter rye were significantly affected by the level of N fertilizer but not by biochar. For N uptake of oil radish an interaction effect was observed for biochar and N fertilizer. Without applied fertilizer, 39% higher N uptake was found in the presence of biochar, accompanied by higher soil NH4+ content and elevated cumulative CO2 emissions. At 130% of the recommended fertilizer level, 16% lower N uptake and lower cumulative N2O emissions were found in the biochar-mediated treatment. No significant change in abundance of microbial groups and nosZ gene were observed. Our results highlight that biochar can have a greenhouse gas mitigation effect at high levels of N supply and may stimulate nutrient uptake when no N is supplied.