Nickel-based superalloy Nimonic 80A with various carbon contents has been developed. Various strengthening mechanisms of carbon were analyzed by optical microscope, X-ray diffraction, scanning electron microscope equipped with energy dispersive spectroscopy and transmission electron microscope. Detailed microstructural analysis revealed that with the increase of carbon content from 0.01% to 0.10%, both the lattice parameters of γ′ and γ phases decreased, and the misfit δ between the coherent γ′ and γ phases increased slightly from 0.289% to 0.317% after full heat treatment at 1070°C/8h, A.C.+700°C/16h, A.C. The diameter of spherical γ′ phase decreased from 22.24nm of 0.01%C alloy to less than 15.76nm of 0.06%C and 0.10%C alloys. Cr 23 C 6 carbide precipitated at the grain boundary in the alloys with carbon content higher than 0.06%, and when carbon content increased to 0.10%, Cr 23 C 6 carbide had an orientation relationship with the γ matrix: [001¯] Cr 23 C 6 [001¯]γ matrix and(100) Cr23C6 (100) γ matrix, which can enforce the grain boundary strength. The growth of room temperature tensile strength with the increase of carbon content was primarily due to the coherent strain strengthening and the precipitate strengthening of Cr 23 C 6 carbide. After stress-rupture test at 750°C/310MPa, the misfit δ increased from 0.182% to 0.237% with the increase of carbon content, but the values were lower than those after full heat treatment. The precipitate of blocky Cr 23 C 6 in 0.10%C alloy tended to become spherical and dislocation lines were found in the γ matrix channels between γ′ phases, the combination of the dislocation strengthening and precipitate strengthening of Cr 23 C 6 carbide at grain boundary was beneficial to the stress-rupture properties.