Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack.