Abstract: Conventional microbead biofilters are simple and reliable in form, but they possess certain limitations due to the thickness of the filter. Excessive filter thickness can cause channeling along the wall and even moderate to severe blockage. Water channeling detrimentally decreases the residing time of the reactants, thus leading to decreased nitrification activity. Sequencing microbead biofilter, in which the media keeps continuous “up and down” movement, is developed on the basis of the traditional microbead filters. The breaker bars are positioned in the bead bed volume in order to generate a relative displacement between microbeads, which enable the biofilter obtain the effect of self-cleaning. The investigation of effect of media layer reciprocating motion on nitritification efficiency and kinetics of this type of filter as a purpose, a pilot scale experiment was carried out in a recirculating aquaculture system. The experimental biofilter was divided into two chambers, designated A-chamber and B-chamber. The water flow through the filtration system was proceed by the electric control valve and water level switch in an alternating fashion, regularly switching between the vessels to enter the A-chamber or B-chamber. Each chamber of the experimental filter was 30 cm in diameter and 50 cm height. The filter used expanded polystyrene beads (microbead) approximately 3.0 mm in diameter, with a density of 28 kg/m3 and a specific surface area of 1160 m2/m3. The microbead packing layer was highly filled at 0.26 m, exhibiting a microbead packing volume of 0.037 m3. The filter used a pump to carry water, while the water trickles down the microbead packing due to gravity. The RAS test system consisting of a 1.3 m3 culture tank, a particle trap, swirl separators, a pump sump, a reuse pump, a sequencing microbead biofilter, and an air diffuser. The bulk water was pumped from a sump to the tested filter after removing solid wastes by the swirl separator, and then returned to culture tank. The total water volume of the system was about 1.6 m3. The reciprocating motion was suspended for six days. Then resume movement of the media and compare the ammonia removal rate and nitritification kinetics in two operation conditions. The result revealed that the movement enhanced the treatment performance greatly. Six days later, when the reciprocating motion was suspended, the ammonia removal efficiency exhibited a gradual decline of 27.1%. Then, the movement of the media was resumed. As a result biological filter ammonia removal efficiency increased rapidly to the level before the motion stops after two days. The results of nitritification kinetics indicate that the media movement changed the kinetics characteristics of filter. Compared with that in stationary state, the threshold value from zero-order to half-order kinetics decreased significantly. The media reciprocating movement and the cutting of the flow greatly improved the hydraulics on the surface of media the mass transfer efficiency. Thereby, the biofilm activity and nitrification the efficiency increased correspondingly.