Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD.