Surface-layer proteins (SLPs) have been detected in all Clostridium difficile strains and play a role in adhesion, although an involvement in the inflammatory process may also be supposed, as they cover the bacterial surface and are immunodominant antigens. The aim of this study was to evaluate the immunomodulatory properties of SLPs obtained from hypervirulent and epidemic (H/E) or non-H/E C. difficile strains, to try to determine whether they contribute to hypervirulence. SLPs were purified from H/E PCR ribotype 027 and 001 and non-H/E PCR ribotype 012 C. difficile strains, and the ability to modulate these properties was studied in human ex vivo models of monocytes and monocyte-derived dendritic cells (MDDCs). The results indicated that SLPs were able to induce immunomodulatory cytokines [interleukin (IL)-1β, IL-6 and IL-10] in monocytes. SLPs induced maturation of MDDCs, which acquired enhanced antigen-presenting activity, a crucial function of the mature stage. SLP-primed MDDCs expressed high levels of IL-10, an important regulatory cytokine. No significant differences were found in the activation induced in monocytes and MDDCs by SLP preparations from H/E and non-H/E strains. Overall, these findings show an important role for SLPs in modulation of the immune response to C. difficile. However, SLPs from H/E strains did not show a specific immunomodulatory pattern compared with SLPs from non-H/E strains, suggesting that SLPs are not involved in the increased severity of infection peculiar to H/E strains.