We have demonstrated a portable microfluidic cell culture system with multi-gas (CO2 and O2) incubation which we can cultivate under hypoxia without bulky peripheral apparatus such as gas tanks, regulators, and flow controllers. The system contains a chip of 26 mm × 48 mm which is capable to diffuse CO2 and absorb O2 through a gas-permeable wall of nested media reservoir. The media was water-jacketed with aqueous solution containing 0.8 M sodium bicarbonate as CO2 supply and 1M sodium ascorbate as oxygen scavenger. The partial CO2 pressure (pCO2) in media reservoir stabilized at least 10.2% ± 0.11% for at least 72 hours. The partial O2 pressure (pO2) in the media reservoir decreased to 4.2%. Portable on-chip hypoxic culture of SV40-T2 cells for 72 h was also demonstrated.