Cadmium-induced nephrotoxicity has been one of the major concerns for public health over the past century. Lipid peroxidation is a principal mechanism in its pathological process. Atmospheric pressure-MALDI mass spectrometry imaging (AP-MALDI MSI) enables direct mapping of lipids in the biological tissue sections. Considering the spatial visualization of lipids on mouse kidney tissues with acute cadmium toxicity is lacking, this study dedicates to filling the gap by using AP-MALDI MSI. Of the tested matrices, the optimized matrix for labeling lipids was 2,5-dihydroxyacetophenone (DHAP). A set of lipids including phosphatidylcholines (PC), phosphatidylglycerol (PG), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidic acid (PA), triglyceride (TG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), etc. were identified and visualized. Accordingly, PC, PG, LPC, SM, PA and TG were down-regulated while PE and PI were up-regulated in the renal cortex or medulla regions in kidney tissues of the mouse with acute cadmium toxicity. Such in situ locations of lipids on mouse kidney tissues with acute cadmium toxicity could help discover tissue-specific nephrotoxic biomarkers and provide new insights into its renal toxicological mechanism.