The physiological oxygen levels for several mammalian cell types in vivo are considered to be hypoxic (low oxygen tension), but the vast majority of in vitro mammalian cell culture is conducted at atmospheric oxygen levels of around 21%. In order to understand the impact of low oxygen environments on cells, oxygen levels need to be regulated during in vitro culture. Two common methods for simulating a hypoxic environment are through the regulation of gas composition or chemical induction. Chemically mimicking hypoxia can have adverse effects such as reducing cell viability, making oxygen regulation in cell culture chambers crucial for long-term culture. However, oxygen-regulating cell culture incubators and commercial hypoxia chambers may not always be a viable option due to cost and limited customization. Other low-cost chambers have been developed, but they tend to lack control systems or are fairly small scale. Thus, the objective of this project was to design and develop a low-cost, open-source, controllable, and reproducible hypoxia chamber that can fit inside a standard cell culture incubator. This design allows for the control of O 2 between 1 and 21%, while maintaining CO 2 levels at 5%, as well as monitoring of temperature, pressure, and relative humidity. Testing showed our hypoxia chamber was able to maintain CO 2 levels at 5% and hypoxic O 2 levels at 1% and 5% for long-term cell culture. This simple and easy-to-manufacture design uses off the shelf components, and the total material cost was $832.47 (USD).