The development of mitochondria during seed germination is essential for plant growth. However, the developmental process is still poorly understood. Temperature plays a key role in soybean germination, and in this study we characterized the mitochondrial ultrastructure and proteome after imbibition at 22, 10 and 4C for 24h. The mitochondria from the soybean seed axis can be divided into light and heavy mitochondria by Percoll density gradient centrifugation. The axes imbibed at 4C mainly contained light mitochondria, which had lower levels of specific mitochondrial enzymes and oxidative phosphorylation activity. In contrast, the axes imbibed at 22C mainly contained heavy mitochondria, which exhibited higher metabolism. Electron microscopy revealed that mitochondria in the axes imbibed at 4C had a poorly developed internal membrane system with few cristae, while the mitochondria in the axes imbibed at 22C developed more normally. Furthermore, we compared the axis mitochondrial proteomes during imbibition at different temperatures. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). Proteins involved in mitochondrial metabolites including malate dehydro-genase (tricarboxylic acid cycle enzyme), putative ATP synthase subunit (oxidative phosphorylation complex subunits), mitochondrial chaperonin-60 (heat shock protein), arginase (urea cycle enzyme) and mitochondrial elongation factor Tu (mitochondrial genome transcript enzyme) were identified. The reduced expression of these proteins might not support normal mitochondrial metabolism. We conclude that chilling during imbibition causes mitochondrial damage at both ultrastructural and metabolic levels.