ABSTRACTDuring host cell invasion,Shigellaescapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with the autophagy process to targetShigellafor destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to studyShigellaseptin cage entrapment in its near-native state. Quantitative cryo-SXT showed thatShigellafragments mitochondria and enabled visualization of X-ray-dense structures (∼30 nm resolution) surroundingShigellaentrapped in septin cages. Using Airyscan confocal microscopy, we observed lysine 63 (K63)-linked ubiquitin chains decorating septin-cage-entrappedShigella. Remarkably, septins and K63 chains are present in separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live-cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy ofShigella. Together, these findings demonstrate how septin-cagedShigellaare targeted for autophagy and provide fundamental insights into autophagy–cytoskeleton interactions.