TcdB is a potent cytotoxin produced by pathogenic Clostridioides difficile that inhibits Rho GTPases by mono‐glucosylation. TcdB enters cells via receptor‐mediated endocytosis. The pathogenic glucosyltransferase domain (GTD) egresses endosomes by pH‐mediated conformational changes, and is subsequently released in an autoproteolytic manner. We here investigated the uptake, localization and degradation of TcdB. TcdB colocalized with lysosomal marker protein LAMP1, verifying the endosomal‐lysosomal route of the toxin. In pulse assays endocytosed TcdB declined to a limit of detection within 2 hr, whereas the released GTD accumulated for up to 8 hr. We observed that autoproteolytic deficient TcdB NXN C698S was degraded significantly faster than wildtype TcdB, suggesting interference of TcdB with lysosomal degradation process. In fact, TcdB reduced lysosomal degradation of endosome cargo as tested with DQ‐Green BSA. Lysosomal dysfunction was accompanied by perinuclear accumulation of LAMP1 and a weaker detection in immunoblots. Galectin‐8 or galectin‐3 was not recruited to lysosomes speaking against lysosome membrane damage. Changes in the autophagosomal marker LC3B suggested additional indirect effect of lysosomal dysfunction on the autophagic flux. In contrast to necrotic signaling induced in by TcdB, lysosomal dysfunction was not abolished by calcium channel blocker nifedipin, indicating separate cytopathogenic effects induced by TcdB during endo‐lysosomal trafficking.