Plant hormones function to coordinate plant growth and development. While the plant hormones, mainly auxin and cytokinin, are exogenously added to various plant tissue cultures, their effects on the organogenesis are apparent, but little is known concerning the molecular mechanisms by which they function in cultured cells. Rice, as a model plant in monocots, is also suitable to tissue culture studies. Here, we used four types of regeneration mediums with different relative concentrations of cytokinin and auxin for rice callus differentiation, the calli at different differentiation stages were collected for proteomic analysis. 2-dimensional electrophoresis revealed that 213 protein spots significantly differentially expressed during callus differentiation under different hormone conditions. By using mass spectrometry, 183 differentially expressed protein spots were identified to match 157 unique proteins. Most of these differential proteins were cellular/metabolic process-related proteins, whose different expression patterns may be correlated with the cytokinin and auxin regulation. Several hormone-related proteins were prominently featured in differentiated calli as compared with the initiated calli, such as alpha-amylase isoforms, mannose-binding rice lectin, putative dehydration stress-induced protein, cysteine endopeptidase and cystatin. All these results provide a novel insight into how the two plant hormones effect the callus differentiation in rice on the proteomic level.