The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.