True-color image of Mars is important for mineral analysis. The working light source of the Mars exploration multi-spectral camera is different from the laboratory calibration light source, so that the commonly used RGB calibration method has a large color difference in correcting the on-orbit images. After analyzing the influence of light source lightness and relative spectral distribution on the camera output values, their influences are considered separately in the calibration. A new color calibration method in the luminance-independent chromaticity space is proposed. The RGB color components of multi-spectral camera are normalized and converted to chromaticity values, and the polynomial calibration model in chromaticity space is compared with the RGB method. The experimental results showed that the average value of CIE DE2000 color differences after color calibration was 4.32, which was 0.96 less than that of RGB method. When the calibration matrix was used to correct the outdoor scene images, the corrected color difference gain because of the change of imaging light source was reduced by 0.54 compared to the RGB method. The color calibration method and the calibration data in this paper provide theoretical support for the color correction of the Mars multi-spectral camera on-orbit images.