The phosphate-dependent transition between enzimatically inert dimers into catalytically-capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated Glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model, consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop L321RFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 in mouse) is here proposed as an important down-regulator of super-oligomer assembly and protein activation. BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), a known glutaminase inhibitor, completely disrupted the higher-order oligomer, explaining its allosteric mechanism of inhibition via tetramer-stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the more active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone super-active GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism.