Global warming and eutrophication of water bodies are driving the increase in cyanobacterial blooms, which produce toxins such as cylindrospermopsin (CYN). This compound has multiple toxic effects, and following CYN exposure, its distribution in the body varies, particularly in organs such as the liver and kidneys, suggesting its potential for bioaccumulation in key tissues. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) enables visualization of the spatial distribution of a wide range of molecules. In this study, using MALDI-MSI, a new method was developed and optimized for the detection of CYN, and its quantitative spatiotemporal distribution was analyzed for the first time in intestinal samples from rats orally exposed to this toxin (500 μg/kg body weight) and sacrificed 0, 2, 4, 6 and 24 h after exposure. Furthermore, the impact of CYN on the intestinal lipid profile was evaluated. The method was validated in terms of linearity, sensitivity, and precision, measuring CYN in mimetic tissue sections at different concentrations (1–100 ppm), allowing its successful application to visualize CYN distribution in rat intestines. The results revealed alterations in different lipid families involved in the inflammatory response, increased oxidative stress, and progressive damage to the integrity of the cell membrane.