Clostridium difficile is a major causative agent of antibiotic-associated diarrhea and has become the most common pathogen of healthcare-associated infection worldwide. The pathogenesis of C. difficile infection (CDI) is mediated by many factors such as colonization involving attachment to host intestinal epithelial cells, sporulation, germination, and toxin production. Bacterial cell surface components are crucial for the interaction between the bacterium and host cells. C. difficile has two distinct surface layer proteins (SLPs): a conserved high-molecular-weight SLP and a highly variable low-molecular-weight SLP. Recent studies have shown that C. difficile SLPs play roles not only in growth and survival, but also in adhesion to host epithelial cells and induction of cytokine production. Sequence typing of the variable region of the slpA gene, which encodes SLPs, is one of the methods currently used for typing C. difficile. SLPs have received much attention in recent years as vaccine candidates and new therapeutic agents in the treatment of C. difficile-associated diseases. Gaining mechanistic insights into the molecular functions of C. difficile SLPs will help advance our understanding of CDI pathogenesis and the development of vaccines and new therapeutic approaches. In this review, we summarize the characteristics and immunological roles of SLPs in C. difficile.