An improved two-dimensional blue native/SDS polyacrylamide gel-electrophoresis (BN/SDS-PAGE) followed by nano-HPLC-ESI-MS/MS was used to study thylakoid membrane protein complexes between a low chlorophyll b rice mutant (Oryza sativa L. Zhenhui 249Y) and its wild type (Oryza sativa L. Zhenhui 249W). The PSⅠ-LHCⅠ super-complexes, LHCⅠ-less PSⅠ, ATP synthase, Cytochrome b6f, CP43-less PSⅡ, trimeric and monomeric LHCⅡ were resolved. 24 proteins, which belong to the subunits of four major photosynthetic apparatus were identified. Comparative study indicated that the mutant had moderately decreased amount of LHCⅡ, greatly decreased amount of LHCⅠ, and increased amount of PSⅡ reaction center and ATP synthase. Western blotting verified BN/SDS-PAGE analysis. These results provided clues about molecular foundation of higher photochemical efficiency and light stability of the mutant, and showed that the improved BN/SDS-PAGE is suitable for not only membrane protein complex separation, but also for comparative study of complex subunits in different physiological condition or between mutant and its wide type.