As an enteric pathogen, Toxoplasma gondii must cross the intestinal mucosal barrier (IMB) to reach the intestinal epithelium to invade host cells. The IMB is a physicochemical barrier against invading pathogens and noxious substances. It is comprised of a mucus layer produced and secreted by goblet cells, an adherent glycocalyx on the surface of enterocytes, and epithelial tight junctions. Specifically, the mucus layer is an adhesive structure made up of large glycoproteins called mucins. Mucin 2 (MUC2) is the major gel-forming mucin in intestinal mucus. Additionally, the mucus layer is replete with antimicrobial peptides (AMPs), including cathelicidins such as LL-37. While Toxoplasma bradyzoites are major mediators of intestinal infection in a new host, their interactions with the IMB are unknown. Here, using immunofluorescence assays (IFA), we show that, unlike tachyzoites that remained trapped in the mucus layer secreted by human goblet cells HT29-MTX-E12 6 hours post-infection, bradyzoites were able to cross this barrier and invade these cells within one hour. By 24h, some of these parasites have divided and/or differentiated into tachyzoites. Furthermore, MUC2 and LL-37 show no antiparasitic effects on bradyzoites, rather parasites pre-treated with these compounds exhibit increased infectivity, as determined by IFA and plaque assays. Together, our findings indicate that bradyzoites possess unique factors that promote their survival in the intestinal mucus layer and subsequent invasion of intestinal epithelial cells. Such molecules may serve as novel therapeutic and diagnostic targets in the fight against toxoplasmosis.