Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research. •Cross-reactive monoclonal antibody 1A4B-6 protects mice prophylactically from lethal Venezuelan equine encephalitis virus challenge.•1A4B-6 inhibits virus growth in vitro at a pre-attachment step after virions are incubated at 37oC and inhibits virus-mediated cell fusion.•Amino acid residue N100 in the fusion loop of E1 protein is critical for binding of 1A4B-6.