Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an azide-and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enabled imaging of sphingolipids and their interactions with proteins in the membrane of intracellular organelles with a spatial resolution of 10-20 nm. Because sphingolipids accumulated efficiently in pathogens we used sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allowed us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm. The Supporting Information is available free of charge on the ACS Publications website. Supplementary Figures S1-S23. Supplementary Movie 1: 10x ExM SIM z-stack of Hela229 cells infected with Chlamydia trachomatis for 24 h, fed with α-NH2-ω-N3-C6-ceramide, fixed, permeabilized and stained with DBCO-Alexa Fluor 488. Chlamydia are clearly located at the inclusion membrane. Scale bar, 10 µm.