Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses remain to be fully defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy appears to involve penetration of the colonic mucus barrier, to reach the underlying epithelium. To better define these interactions, we grew colonoids under air-liquid interface (ALI) conditions, producing a thick mucus layer that mimicked in vivo mucus composition and glycosylation. C. rodentium’s penetration of ALI mucus was dramatically enhanced upon exposure to sialic acid, in concert with the secretion of two autotransporter (SPATE) proteins, Pic and EspC. Despite Pic being a class II SPATE, and already recognized as a mucinase, it was EspC, belonging to the class I SPATE family, and not previously shown to possess mucinase activity, that degraded the ALI-derived mucus. Confirming this finding, E. coli DH5 carrying a plasmid that expresses C. rodentium-derived EspC was able to degrade the mucus. Notably, recombinant EspC alone also displayed mucinolytic activity in a dose-dependent manner. Collectively, our results reveal the utility of ALI-derived mucus in modeling microbe-host interactions at the intestinal mucosal surface, as well as identify EspC as an atypical Class I SPATE that shows significant mucinolytic activity towards ALI-derived mucus.