Abstract Biological nitrification inhibition (BNI) of Brachiaria humidicola has been attributed to nitrification‐inhibiting fusicoccanes, most prominently 3‐ epi ‐brachialactone. However, its release mechanism from B. humidicola roots remains elusive. Two hydroponic experiments were performed to investigate the role of rhizosphere pH and nutritional N form in regulating 3‐ epi ‐brachialactone release by B. humidicola and verify the underlying release pathway. Low rhizosphere pH and NH 4 + nutrition promoted 3‐ epi ‐brachialactone exudation. However, the substitution of NH 4 + by K + revealed that the NH 4 + effect was not founded in a direct physiological response to the N form but was related to the cation‐anion balance during nutrient uptake. Release of 3‐ epi ‐brachialactone correlated with the transmembrane proton gradient ΔpH and NH 4 + uptake ( R 2 = 0.92 for high ~6.8 and R 2 = 0.84 for low ~4.2 trap solution pH). This corroborated the release of 3‐ epi ‐brachialactone through secondary transport, with the proton motive force (ΔP) defining transport rates across the plasma membrane. It was concluded that 3‐ epi ‐brachialactone release cannot be conceptualized as a regulated response to soil pH or NH 4 + availability, but merely as the result of associated changes in ΔP.