It has been reported that ammonia produced by glutaminolysis activates the HIF-1 pathway in several types of cancer cells, but the underlying mechanisms remain unclear. In this study, the effects of ammonia on the activation of HIF-1 pathway and glycolysis in MDA-MB-231 breast cancer cells were investigated and the underlying mechanisms involved were elucidated. The results showed that NH 4 Cl concentration-dependently increased the protein level of HIF-1α and enhanced the transactivation activity of HIF-1 in MDA-MB-231 cells. In addition, NH 4 Cl increased the expression of GluT1 and LDHA and promoted aerobic glycolysis by activating the HIF-1 pathway. Further study revealed that NH 4 Cl increased the mitochondrial ROS level and decreased the cellular Fe2+ level in MDA-MB-231 cells. Activation of the HIF-1 pathway induced by NH 4 Cl was inhibited by addition of the antioxidant NAC or the NADPH oxidase (NOX) inhibitor apocynin, indicating the involvement of the NOX-induced ROS generation. When MDA-MB-231 cells were treated with NH 4 Cl, the oxygen consumption of cells increased, followed by the decreased mitochondrial membrane potential and cellular ATP level, indicating the uncoupling of mitochondria. In conclusion, NH 4 Cl activated the HIF-1 signaling pathway and promoted aerobic glycolysis in MDA-MB-231 cells, likely through the promotion of mitochondrial ROS release and mitochondrial uncoupling. • NH 4 Cl activated HIF-1-dependent aerobic glycolysis in MDA-MB-231 cells. • NH 4 Cl promoted NOX-dependent ROS generation in MDA-MB-231 cells. • NH 4 Cl induced mitochondrial uncoupling in MDA-MB-231 cells. [ABSTRACT FROM AUTHOR]