Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Surface layer proteins (SLPs) are the most abundant surface localised proteins expressed by C. difficile. The aim of this study was to examine the humoral immune response to C. difficile SLPs and its potential role in protection from C. difficile associated diarrhoea (CDAD). Serum antibodies to SLPs from C. difficile were measured by ELISA in a cohort of 146 patients (55 patients with CDAD, 34 asymptomatic carriers, and 57 controls). No significant difference was detected in serum IgM, IgA or IgG antibody levels between cases, carriers or control groups at any of the time points tested. However, patients with recurrent episodes of C. difficile diarrhoea had significantly lower IgM-anti-SLP levels than patients with a single episode on days 1, 3, 6 and 9 (p = 0.05, p = 0.009, p = 0.02, p = 0.049). The adjusted odds ratio for recurrent diarrhoea associated with a low day 3 serum IgM anti-SLP antibody level was 24.5 (95% confidence interval; 1.6-376.3). Further studies which examine the specific anti-SLP antibody responses to the colonising strain are warranted to determine if immune responses to C. difficile SLPs play a role in protection from CDAD.