The deformation microstructures of two Ni-based superalloys with different Co contents after creep tests at 650 °C/630 MPa, 725 °C/630 MPa and 760 °C/630 MPa were investigated by transmission electron microscopy (TEM), in order to study the influence of temperature and stacking fault energy (SFE) on the creep deformation mechanisms. The results show that the improvement in temperature enhances the creep mechanism transition from stacking faults to micro-twinning for the experimental single crystal alloys, suggesting that the formation of micro-twins is dependent on temperature. Moreover, increasing Co content as well as lowering SFE allow stacking faults or micro-twins to extend through the γ matrix and γ′ precipitates, which improve the creep resistance and prolong the creep life of the alloys.