The transition in cellular metabolism supporting cancer cell proliferation generates an acidic extracellular pH (pHe) environment to provide survival advantage over non-malignant cells(1). The intrinsic intracellular buffering capacity (βI) dictates the intracellular pH (pHi) response to acidic pHe(2). Additionally, pHi regulatory mechanisms establish the pHi homeostasis influencing cellular processes contributing to oncogenesis(3). Hypoxia exacerbates acidosis exerting selection pressure that promote and sustain oncogenesis(4). We hypothesized that altered acidic microenvironment influences pHi regulation in ovarian cancer cells (SKOV3). We investigated the change in buffering capacity of the cancer cells and correlated the pH recovery rate to attain steady state pHi in response to an altered pHe. The pHi change was mapped to understand the proton dynamics of pHi regulation in altered pHe. Cells were cultured at 6.3-6.5 or 7.2-7.4 pHe in parallel with either normal conditions (0.21 atm) or at 0.029 atm PO2 mimicking in vivo tumour environment. The measured pHi distribution was mapped across the cSNARF(pH-sensing dye) loaded cells, using wide-field microscope in live cell setup. The acid dumping capacity of the cell was assessed by acutely loading them with acid and monitoring the subsequent pHi recovery towards initial levels in respective pHe. The pHi recovery data points were fitted through the two limbs of a segmented linear regression curve. The differences between the slopes separated by breakpoints described an abrupt change in pHi recovery in altered pHe. The βI of cells(N>7) drifted from 0.2 to 0.6(mM/pH) when switching from physiologically pHe(7.4) to 6.3 at atmospheric PO2 . The pHi recovery was achieved by multiple acid extruding mechanisms. A pulse of 20mM NH4+, produced gradual pHi recovery of 0.3 & 0.5(pH/minutes) for pHe 7.4 & 6.3 respectively. The recovery slowed to 0.08pH/minutes and 0.09pH/minutes after 18,16 minute breakpoints for pHe 7.4 and 6.3 respectively. Interestingly the slopes of linear regression lines during initial 2 minutes of pHi recovery were significantly (p<0.005) different. The centre of the cells reported a predominant pHi of 6.99 for cells in 6.3 pHe and 7.35 in 7.4 pHe at 0.21 atm PO2 . The pHi distribution manifested differentially, contributing to unique steady state pHi in altered pHe. The extent of βI of ovarian cancer cells was dependent on altered microenvironmental proton dynamics. Our results highlighted that cancer cells adapt to changes in their microenvironment favouring unique pathophysiological processes through regulation of pHi. It is important to understand how proton dynamics influence transformation of non-malignant cells, to develop potential therapeutic strategies. Further investigations will reveal new avenues to meet the challenge of targeting tumour metabolism.