Abstract: Lipids are one of the most important biomolecules, where they serve as building blocks for celluar membranes and involve in signal transduction and energy storage. The molecular imaging of lipids is critical for understanding complex biological processes, especially under pathological conditions. Spinal cord injury (SCI) always leads to a severe insult motor and sensory function impairment. The direct physical damage to spinal cord will lead to secondary injury consisted of uncontrollable oxidative stress and sever neuroinflammation, subsequently causes lipid peroxidation. The oxidative stress and lipid peroxidation in SCI result in alterations of lipid metabolism and homeostasis, thus induce the changes and heterogeneous spatial distribution of lipids. Therefore, mapping the lipid distribution in injured spinal cords is necessary for understanding the pathological microenvironment of SCI. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a powerful imaging tool for in situ visualization of numerous biomolecules simultaneously. To improve the matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) performance of lipids, developing new matrices is urged. Our previous studies have shown that the matrix of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) exhibits excellent performance for detecting hydrophobic proteins and peptides, which has good co-crystallization performace and increased hydrophobicity. Herein, CHCA-C3 was applied for MALDI-MSI of lipids after SCI, and the changes and spatial distribution of lipids at different times after SCI were analyzed. Compared with CHCA, CHCA-C3 shows profound improvement in lipid detection, that more MS peaks are obtained with higher intensity. Using CHCA-C3, 251 MS peaks are observed in MS spectra, and 25 of them are identified as lipids by searching against database. Moreover, the difference of lipid distribution in injured spinal cords at various time after SCI was investigated. And four lipids, including cholesterol ester CE (20:4), phosphatidylcholine PC (32:1), PC (34:0) and PC (40:0), show significant difference in the spatial distribution at 12 h (acute stage), 3 day (subacute stage) and 7 day (subacute stage) after SCI. Subsequently, the segmentation model (SM) and principal component analysis (PCA) were performed to comprehensive analyzing the 251 MS peaks, and obvious differences are found at spectra obtained from injured spinal cord at different injured phases. In conclusion, this research not only expands the application of CHCA-C3 in MALDI-MSI, but also provides the changes and spatial distribution of lipids in SCI. And the result provides the in-depth understanding of lipidomic changes in SCI, as well as supports the development of new therapeutic strategies for SCI treatment.