Oral administration of Clostridium butyricum as probiotic is increasingly gaining importance in the treatment of diarrhea and the improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The objective of this study was to analyze the mechanisms involved in C. butyricum induction of the toll-like receptor (TLR) signaling. Knockdown of myeloid differentiation primary response protein 88 (MyD88) expression using small interfering RNA in this manner did not affect C. butyricum-induced elevated levels of nuclear factor κB (NF-κB), interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α), suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6, and TNF-α was evident in the absence of TLR2 expression, implying the need for TLR2 in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells, which adds to our understanding of the molecular mechanisms of this probiotic action on gut epithelium.