Background: Previous research suggests that low serum concentrations of the third component of complement (C3) are associated with both the susceptibility to infectious agents such as Borrelia burgdorferi and the development of glomerular disease. We hypothesized that low levels of C3 are associated with the coincident occurrence of B. burgdorferi infection and glomerulonephritis in Bernese Mountain dogs. Objectives: The aims of this study were to evaluate the serum concentration of C3 in Bernese Mountain dogs with and without antibodies against B. burgdorferi and to compare this concentration with that of healthy control dogs. Methods: Eighty-three clinically healthy Bernese Mountain dogs and 46 control dogs were included. Antibodies against B. burgdorferi were determined using an ELISA with a whole cell sonicate as antigen. Results were confirmed using Western blot. C3 was measured using a single radial immunodiffusion test. Results were reported as the percentage concentration of C3 compared with that in pooled preserved canine serum (100% C3 concentration). Results: Median C3 concentration was 128.5% in Bernese Mountain dogs with antibodies against B. burgdorferi, 133.5% in B. burgdorferi-negative Bernese Mountain dogs, 87.8% in positive control dogs, and 102.2% in negative control dogs. Within Bernese Mountain and control groups, C3 was lower in dogs with antibodies against B. burgdorferi compared with those without. Percentage concentration of C3 was higher in healthy Bernese Mountain dogs compared with control dogs. Conclusion: Low C3 concentration is not an explanation for the high prevalence of B. burgdorferi infections and glomerular disease in Bernese Mountain dogs.