An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.