Biological nitrification inhibitors (BNIs) are released from plant roots as exudates to repress nitrifier activity in agricultural soils, and this can improve nitrogen (N) recovery from fertilizer and enhance the N-use-efficiency (NUE). This review summarizes the current understanding of the regulatory mechanisms of BNIs release from roots of plants, such as Brachiaria humidicola (pasture grasses), Sorghum bicolor (hybrid sorghum) and Oryza sativa (paddy rice). BNIs can be categorized as hydrophilic- and hydrophobic-BNIs. Root systems can rapidly release hydrophilic-BNIs when NH4+ is present in rhizosphere in combination with low pH, which is associated with the activation of plasma membrane H+-ATPase. Since plasma membrane H+-ATPase is responsible for the establishment of membrane potential and generation of proton motive force for the secondary transport of various substances. The BNIs release may probably occur through the voltage-gated anion channels by the membrane potential variation or via secondary transporters, most likely MATE transporters, powered by the proton motive force. In addition, ATP-binding cassette (ABC) transporters may be also involved in the active efflux of hydrophilic-BNIs. On the contrary, the release of the hydrophobic BNIs, such as sorgoleone, from plant roots may be mediated by the vesicle traffic process and/or exocytosis. In addition, the possible effects of various environmental factors on the BNIs release in soils have been discussed. Future research should focus on the identification of the corresponding BNIs transporters in plants, and this may be helpful for the application of BNI crops in the agricultural practice via breeding and genetic modification.