In order to understand the mechanism of neckpanicle elongation in protein level, we used ‘Changxuan 3S’ and ‘Peiai 64S’ as experimental materials. The proteins in the uppermost internodes were separated on the 2 day before heading by using solidphase pH gradient twodimensional gel electrophoresis and mass spectrometry. A twodimensional gel electrophoresis map (2DE) with good resolution and reproducibility was obtained. And the different proteins were studied. The results showed that: (1) two dimensional gel electrophoresis maps with good resolution and reproducibility were obtained. (2) 40 differential protein spots were selected from the 2DE map and analyzed by MALDITOF/TOFMS peptide mass fingerprinting, and 27 of them were identified successfully. Compared with ‘Peiai 64S’, there are 17 upregulated and 10 downregulated proteins in ‘Changxuan 3S’. (3) According to their functions, these differential proteins can be divided into six categories, mainly including cell metabolism relative proteins and cell wall reconstruction proteins. In addition, these discrepant proteins may be interrelated to the vigorous elongation of the internodes at heading stage of ‘Changxuan 3S’, especially the proteins related to cell wall reconstruction and cell elongation. (4) The protein spot 2, 7, 8, 24, 35 and 36 were tested by qPCR. The expression levels of 2 (Os10g08550), 7 (Os12g42876) and 8 (Os01g55830) in ‘Changxuan 3S’ were significantly lower than that in ‘Peiai 64S’, while the expression levels of 24 (Os06g48760), 35 (Os05g25850) and 36 (Os07g42300) were significantly higher than those of ‘Peiai 64S’. The qPCR results were consistent with the results of protein gel analysis. According to the study, the eui may promote cell division and elongation by regulating the expression of these proteins at heading stage.