The immunomodulator AS101 has been shown to induce cell proliferation and to increase the secretion of a variety of cytokines. In the present study we evaluated the effect of AS101 on the pathogenicity of B. rodhaini infected mice. In order to clarify its mechanism of action we studied the ability of AS101 to activate neutrophils and macrophages, both of which inhibit parasite growth. More specifically, we studied the ability of AS101 to induce secretion of nitric oxide (NO). We found that AS101 protects mice from babesiosis in a time and dose dependent manner. At 10 and 20 μg/injection, two weeks prior to parasites, AS101 significantly increased the number of neutrophils and more than doubled the survival rate of infected mice. Similarly, at these concentrations when injected one month, or at 20 μg, injected 24 h before parasites, AS101 mitigated the course of infection and reduced by half the peak of parasitaemia. At 0.1 μg/ml AS101 induced the secretion of significantly higher levels of NO in vitro than control. This was abrogated by adding the NO synthase inhibitor, NG-monomethyl-L-arginine. In vivo the antiparasitic protection of AS101 was abrogated by another NO synthase inhibitor, aminoguanidine. We found that AS101, partly by elevating levels of NO, can significantly mitigate the course of infection and thus increase survival, and may therefore be proven as an effective antiparasitic drug.