Callus differentiation is a key developmental process for rice regeneration from cells. To better understand this complex developmental process, we used a 2-D gel electrophoresis approach to explore the temporal patterns of protein expression at the early stages during rice callus differentiation. This global analysis detected 60 known proteins out of 79 gel spots identified by MS/MS, of which many had been shown to play a role in plant development. Two new proteins were revealed to be associated with the callus differentiation and have been confirmed by Western blot analysis. The results of proteomics experiments were further verified at the mRNA level using microarray and real-time PCR. Comparison of the differentially expressed protein levels with their corresponding mRNA levels at the two callus early differentiation stages showed a good correlation between them, indicating that a substantial proportion of protein changes is a consequence of changed mRNA levels, rather than post-transcriptional effects during callus differentiation, though microarray revealed more expression changes on RNA levels. These findings may contribute to further understanding of the mechanisms that lead to callus differentiation of rice and other plants as well.