Imaging mass spectrometry (IMS) is a technique for simultaneously acquiring the expression and distribution of molecules on the surface of a sample, and it plays a crucial role in spatial omics research. In IMS, the time cost and instrument load required for large data sets must be considered, as IMS typically involves tens of thousands of pixels or more. In this study, we developed a high-resolution method for IMS data reconstruction using a window-based Adversarial Autoencoder (AAE) method. We acquired IMS data from partial cerebellum regions of mice with a pitch size of 75 μm and then down-sampled the data to a pitch size of 150 μm, selecting 22 m/z peak intensity values per pixel. We established an AAE model to generate three pixels from the surrounding nine pixels within a window to reconstruct the image data at a pitch size of 75 μm. Compared with two alternative interpolation methods, Bilinear and Bicubic interpolation, our window-based AAE model demonstrated superior performance on image evaluation metrics for the validation data sets. A similar model was constructed for larger mouse kidney tissues, where the AAE model achieved high image evaluation metrics. Our method is expected to be valuable for IMS measurements of large animal organs across extensive areas.