The effect of anti-TNF-α treatment in B. burgdorferi infected C3H/He mice treated with ceftriaxone was evaluated in this study. Mice were infected with B. garinii Å218 or B. burgdorferi sensu stricto N40. At two weeks of infection, a group of ten mice was treated with ceftriaxone for five days, while another group received anti-TNF-α simultaneously with ceftriaxone. A third group received anti-TNF-α four weeks after the ceftriaxone treatment and a fourth group received only anti-TNF-α. Ceftriaxone improved joint swelling and decreased B. burgdorferi specific IgG antibody responses. At 14 weeks of infection, B. burgdorferi could not be detected by cultivation or by PCR from tissue samples of any of the animals treated only with ceftriaxone. However, spirochetes grew from the tissue samples of one third of the mice treated with both anti-TNF-α and ceftriaxone, regardless of whether the treatments were received simultaneously or four weeks apart. No loss of plasmids, decreased ceftriaxone sensitivity or virulence rate was found when the activated spirochetes were compared to the bacteria used to infect the mice. All infected control mice and the mice that only received anti-TNF-α were culture positive. These results show that a number of B. burgdorferi infected mice still have live spirochetes which are activated by the anti-TNF-α treatment in their body after treatment with ceftriaxone.