Since the seminal studies of Otto Warburg in the 1920s it has been widely recognized that cancers grow glycolytically even in the presence of oxygen. This generates an abundance of protons in a gradient across most solid tumors with an acidic core and an alkaline rim. Whether and how this proton gradient may also serve in an autocrine fashion on these tumors is unclear. Here we demonstrate that human glioma cells form spheroids which act as a viable three-dimensional tumor model, forming physiologically relevant extracellular pH (pH_e) and cell proliferation gradients. Using fluorescent cell cycle trackers we determined that the rate of cell proliferation is directly dependent on pH_e, and that cells adjust their growth rate according to their position within the pH gradient. We further show that glioma cells sense pH via H⁺-sensitive K⁺ channels that translate changes in pH into changes in membrane voltage. These channels are tonically active and blocked by acidic pH_e, quinine, and ruthenium red. Blockade of this K⁺ conductance either by acidic pH_e or drug inhibition depolarized both glioma cells and tumor spheroids and prevented them from passing through the hyperpolarization-dependent G₁-to-S phase cell cycle checkpoint, thereby inhibiting cell division. In this way, pH_e directly determines the proliferative state of glioma cells.