Serum samples from hunters (n = 440), their hunting dogs (n = 448), and hunters without dog ownership (n = 53) were collected in The Netherlands at hunting dog trials and were tested for antibodies against Borrelia burgdorferi by a whole-cell enzyme-linked immunosorbent assay. Additionally, 75 healthy pet dogs were tested. The results of this study indicate that the seroprevalence among hunting dogs (18%) was of the same order as the seroprevalence among pet dogs (17%) and hunters (15%). The seropositivity of a hunting dog was not a significant indicator of increased risk of Lyme borreliosis for its owner. No significant rise in seroprevalence was found in dogs older than 24 months. This indicated that seropositivity after an infection with B. burgdorferi in dogs is rather short, approximately 1 year. In humans this is considerably longer but is also not lifelong. Therefore, the incidence of B. burgdorferi infections among dogs was greater than that among hunters, despite a similar prevalence of seropositivity among hunters and their hunting dogs. Because no positive correlation was observed between the seropositivity of a hunter and the seropositivity of the hunter's dog, direct transfer of ticks between dog and hunter does not seem important and owning a dog should not be considered a risk factor for Lyme borreliosis.