HIV-1 envelope glycoprotein (Env) interacts with cellular receptors and mediates virus entry into target cells. Blocking Env-receptor interactions represents an effective interventional strategy for developing HIV-1 entry inhibitors. We previously designed a panel of CD4-linker-DC-SIGN (CLD) constructs by fusing the extracellular CD4 and DC-SIGN domains with various linkers. Such CLDs produced by the prokaryotic system efficiently inhibited HIV-1 infection and dissemination in vitro and ex vivo. In this study, following the construction and identification of the most promising candidate with a linker of 8 Gly 4 Ser repeats (named CLD), we further designed an improved form (named CLDmut) by back mutating Cys to Ser at amino acid 60 of CD4. Both CLD and CLDmut were produced in mammalian (293F) cells for better protein translation and modification. The anti-HIV-1 activity of CLD and CLDmut was assessed against the infection of a range of HIV-1 isolates, including transmitted and founder (T/F) viruses. While both CLD and CLDmut efficiently neutralized the tested HIV-1 isolates, CLDmut demonstrated much higher neutralizing activity than CLD, with an IC 50 up to one log lower. The neutralizing activity of CLDmut was close to or more potent than those of the highly effective HIV-1 broadly neutralizing antibodies (bNAbs) reported to date. Findings in this study indicate that mammalian cell-expressed CLDmut may have the potential to be used as prophylaxis or/and therapeutics against HIV-1 infection.