Physiological oxygen levels within the tissue microenvironment are usually lower than 14%, in stem cell niches these levels can be as low as 0–1%. In cell cultures, such low oxygen levels are usually mimicked by altering the global culture environment either by O2 removal (vacuum or oxygen absorption) or by N2 supplementation for O2 replacement. To generate a targeted cellular hypoxic microenvironment under ambient atmospheric conditions, we characterised the ability of the dissolved oxygen-depleting sodium sulfite to generate an in-liquid oxygen sink. We utilised a microfluidic design to place the cultured cells in the vertical oxygen gradient and to physically separate the cells from the liquid. We demonstrate generation of a chemical in-liquid oxygen sink that modifies the surrounding O2 concentrations. O2 level control in the sink-generated hypoxia gradient is achievable by varying the thickness of the polydimethylsiloxane membrane. We show that intracellular hypoxia and hypoxia response element-dependent signalling is instigated in cells exposed to the microfluidic in-liquid O2 sink-generated hypoxia gradient. Moreover, we show that microfluidic flow controls site-specific microenvironmental kinetics of the chemical O2 sink reaction, which enables generation of intermittent hypoxia/re-oxygenation cycles. The microfluidic O2 sink chip targets hypoxia to the cell culture microenvironment exposed to the microfluidic channel architecture solely by depleting O2 while other sites in the same culture well remain unaffected. Thus, responses of both hypoxic and bystander cells can be characterised. Moreover, control of microfluidic flow enables generation of intermittent hypoxia or hypoxia/re-oxygenation cycles. Statement of Significance: Specific manipulation of oxygen concentrations in cultured cells’ microenvironment is important when mimicking low-oxygen tissue conditions and pathologies such as tissue infarction or cancer. We utilised a sodium sulfite-based in-liquid chemical reaction to consume dissolved oxygen. When this liquid was pumped into a microfluidic channel, lowered oxygen levels could be measured outside the channel through a polydimethylsiloxane PDMS membrane allowing only for gaseous exchange. We then utilised this setup to deplete oxygen from the microenvironment of cultured cells, and showed that cells responded to hypoxia on molecular level. Our setup can be used for specifically removing oxygen from the cell culture microenvironment for experimental purposes and for generating a low oxygen environment that better mimics the cells’ original tissue environments.