The final step, and the most crucial for the fish, is to reoxygenate the culture water as it returns to the fish tank. Oxygen is the first limiting factor in recirculating aquaculture systems and with less than the required levels most fish and other aquatic organisms will die in a very short amount of time. It is also critical that biofilters have access to adequate oxygen. Biofilters are homes to nitrifying bacteria, which are aerobic (use oxygen during respiration). Furthermore, nitrification, the conversion of ammonia to non-toxic nitrate by the bacteria, can't occur without the presence of oxygen.

Oxygen can be supplied to the culture water by several methods. An agitator mechanically agitates the surface water, causing oxygen to diffuse into the water as air and water mix. Agitators are commonly used for transporting fish and for emergency aeration in pond culture. A more common method used in small to moderate sized aquaculture systems is to deliver a desired volume of air directly into the water column with an aerator. The most common type of aerator is the regenerative blower. Aerators deliver air through plastic tubing to a diffuser (air stone). Diffusers break air into bubbles to improve the rate of diffusion into the water. The size of the bubbles is dependent upon the size of the pores in the diffuser, the smaller the pores, the smaller the bubbles and the better the rate of diffusion. However, for most applications a medium sized pore is preferred as the smaller the pores, the more pressure and maintenance are required to keep the diffuser operational. Finally, in intensive production systems, for transporting fish or as an emergency back-up, pure oxygen is often injected directly into the water under pressure.

It is estimated that between 600 and 1,000 grams of oxygen is consumed per kilogram (1,000 grams) of feed fed in aquaculture recirculation systems. This estimate includes oxygen consumed by all organisms living in the culture water (i.e., fish, bacteria, protozoa, algae, etc.) as well as the amount of oxygen required for the decomposition of wastes (biological oxygen demand). To be safe, dissolved oxygen levels should be maintained above 5 ppm and no higher than 15 ppm. Oxygen levels should be monitored regularly. A dissolved oxygen meter or chemical test kit is highly recommended.