反硝化反应（英语：denitrification，亦称为反硝化作用、脱氮作用）是指细菌将硝酸盐（NO₃）中的氮（N）通过一系列中间产物（NO₂、NO、N₂O）还原为氮气分子（N₂）的生物化学过程。参与这一过程的细菌统称为反硝化菌或脱氮细菌。

反硝化菌在无氧条件下，以将硝酸盐（NO₃）为电子受体完成呼吸作用（respiration）以获得能量。这一过程是硝酸盐呼吸（nitrate respiration）的两种途径之一，另一种途径是另一类是硝酸异化还原成铵盐（DNRA）。

Denitrification is a microbially facilitated process of nitrate reduction (performed by a large group of heterotrophic facultative anaerobic bacteria) that may ultimately produce molecular nitrogen (N₂) through a series of intermediate gaseous nitrogen oxide products. This respiratory process reduces oxidized forms of nitrogen in response to the oxidation of an electron donor such as organic matter. The preferred nitrogen electron acceptors in order of most to least thermodynamically favorable include nitrate (NO₃), nitrite (NO₂), nitric oxide (NO), nitrous oxide (N₂O) finally resulting in the production of dinitrogen (N₂) completing the nitrogen cycle. Denitrifying microbes require a very low oxygen concentration of less than 10%, as well as organic C for energy. Since denitrification can lower leaching of NO₃ to groundwater, it can be strategically used to treat sewage or animal residues of high nitrogen content. Denitrification allows for the production of N₂O, which is a greenhouse gas that can have a considerable influence on global warming.