At an extremely high Mach number, the regenerative cooling of traditional kerosene cannot meet the requirement of the heat sink caused by aerodynamic heating and internal combustion in a scramjet propulsion system. As a supplement of traditional regenerative cooling, supercritical CO2 is regarded as an effective coolant in severe heating environments due to its excellent properties of heat and mass transportation. In this paper, the heat transfer and flow structure characteristics of regenerative cooling in a rectangular channel using supercritical CO2 are analyzed numerically using a validated model. The effect of heat flux magnitude, nonuniform heat flux, acceleration and buoyancy and flow pattern are considered to reveal the regenerative cooling mechanism of supercritical CO2 in the engine condition of a scramjet. The results indicate that the heat transfer deterioration phenomenon becomes obvious in the cooling channel loaded with relatively high heat flux. Compared with the cooling channels loaded with increased heat flux distribution, the maximum temperature increased for the channel loaded with decreased heat flux distributions. When larger acceleration is applied, a relatively lower wall temperature distribution and higher heat transfer coefficients are obtained. The wall temperature distribution becomes more uniform and the high-temperature region is weakened when the coolants in adjacent channels are arranged as a reversed flow pattern. Overall, the paper provides some references for the utilization of supercritical CO2 in regenerative cooling at an extremely high Mach number in a scramjet.