Treating nitrogen-rich reject water from anaerobically digested sludge with deammonification has become a very beneficial side stream process. One common technique is the one-stage moving bed bioreactors (MBBRs), which in comparison with the other deammonification techniques can be started up without seeding anammox bacteria. This study investigated the impact of biofilm seeding on the start-up of one-stage deammonification MBBRs. Two lab-scale reactors were run in parallel with partial nitritation for 56 days until 11% of the carrier area in one reactor was replaced with fully developed deammonification biofilm to work as the seeding material. The seeded reactor started nitrogen reduction immediately up to a plateau of 1.3 g N m⁻² d⁻¹; after another 54 days on day 110, the reduction significantly increased. At the same time, the non-seeded reactor also started to reduce nitrogen due to deammonification. The development was followed with both nitrogen analyses and fluorescence in situ hybridization analyses. On day 134, the biofilm in both reactors contained>90% anammox bacteria and reached maximum nitrogen removal rates of 7.5 and 5.6 g N m⁻² d⁻¹ in the seeded and non-seeded reactor, respectively. Over 80% of the inorganic nitrogen was reduced. In conclusion, the seeding did not contribute to a shorter start-up time or the achieved anammox enrichment, although it did contribute to a partial, immediate nitrogen reduction. The boundary conditions are the most important factors for a successful start-up in a deammonification MBBR system.