Early stages of mucosal infection are potential targets for HIV-1 prevention. CD4 is the primary receptor in HIV-1 infection whereas DC-SIGN likely plays an important role in HIV-1 dissemination, particularly during sexual transmission. To test the hypothesis that an inhibitor simultaneously targeting both CD4 and DC-SIGN binding sites on gp120 may provide a potent anti-HIV strategy, we designed constructs by fusing the extracellular CD4 and DC-SIGN domains together with varied arrangements of the lengths of CD4, DC-SIGN and the linker. We expressed, purified and characterized a series of soluble CD4-linker-DC-SIGN (CLD) fusion proteins. Several CLDs, composed of a longer linker and an extra neck domain of DC-SIGN, had enhanced affinity for gp120 as evidenced by molecular-interaction analysis. Furthermore, such CLDs exhibited significantly enhanced neutralization activity against both laboratory-adapted and primary HIV-1 isolates. Moreover, CLDs efficiently inhibited HIV-1 infection in trans via a DC-SIGN-expressing cell line and primary human dendritic cells. This was further strengthened by the results from the human cervical explant model, showing that CLDs potently prevented both localized and disseminated infections. This is the first time that soluble DC-SIGN-based bifunctional proteins have demonstrated anti-HIV potency. Our study provides proof of the concept that targeting both CD4 and DC-SIGN binding sites on gp120 represents a novel antiviral strategy. Given that DC-SIGN binding to gp120 increases exposure of the CD4 binding site and that the soluble forms of CD4 and DC-SIGN occur in vivo, further improvement of CLDs may render them potentially useful in prophylaxis or therapeutics.