A high oleic acid rapeseed material M15 (derived from Xiangyou 15 variety) has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy (75%), stress and defense (8.3%), photosynthesis (6.3%), protein metabolism (2.1%) and other functions (8.3%). Then real-time quantitative PCR (qPCR) analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.