The effect of glycine betaine (GB) treatment on chilling injury (CI), cell membrane integrity and membrane fatty acid metabolism in peach fruit during storage were investigated using biochemical and transcriptomic analysis in this study. The results showed that GB treatment alleviated the CI by suppressing lipoxygenase (LOX), phospholipase D (PLD) and lipase gene expression and enzyme activities, resulting in higher levels of unsaturated fatty acids and degree of unsaturation. In addition, this study suggested that GB treatment could inhibit the release of cytochrome C and caspase-3 activity, thus suppressing cell breakdown and contributing to membrane function. Moreover, transcriptome analysis revealed that genes related to fatty acid biosynthesis and desaturation were enhanced, while genes related to fatty acid degradation and activation of caspases were suppressed by GB treatment. Therefore, GB treatment could maintain normal cell membrane structure and function through upregulating membrane fatty acid biosynthesis and desaturation metabolism and downregulating caspase activation to alleviate CI in peaches.