Bothropstoxin-I (BthTX-I) from Bothrops jararacussu snake venom has a predominantly postsynaptic action that is responsible for this toxin´s myotoxicity. However, BthTX-I also has a presynaptic action that is counteracted by Mn2+, a reversible neuromuscular blocker that acts predominantly presynaptically. In this work, we used two nerve-muscle preparations (mouse phrenic nerve-diaphragm - PND and extensor digitorum longus - EDL) to investigate the ability of Mn2+ to protect against the myotoxicity of BthTX-I. The preparations were incubated with Tyrode solution (control), BthTX-I, or Mn2+ alone. BthTX-I (1.4 μM) produced irreversible blockade in both preparations, whereas the blockade by Mn2+ (0.9 mM) was total and reversible in PND but just partially reversible in EDL. Pretreating the preparations with Mn2+ resulted in 100% and 80% protection against BthTX-I-induced blockade, respectively. However, when Mn2+ (0.9 or 1.8 mM) and BthTX-I (1.4 μM) were co-incubated for 30 min before testing, the blockade was faster and sustained. Washing the preparations resulted in complete, sustained recovery in those exposed to 1.8 mM Mn2+ but not to 0.9 mM Mn2+. Morphological analysis showed that the extent of fiber damage by BthTXI (1.4 μM) was 82% (PND) and 68.5% (EDL), and that Mn2+ (0.9 mM) afforded 40% protection in both preparations and reduced the increase in muscle fiber cross-sectional area by 20% and 15%, respectively, compared to BthTX-I alone. Mn2+ (0.9 mM) significantly attenuated the release of creatine kinase by BthTXI. The low creatine kinase activity resulted from a protective action of Mn2+ on the sarcolemma and from direct inactivation of the released enzyme. These results show that Mn2+ prevents membrane disruption by BthTX-I and can protect against the myotoxicity and neurotoxicity caused by this toxin.