HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1(IIIB) entry and replication with EC(50) values of 3.92+/-0.62 and 6.59+/-1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1(KM018) with EC(50) values of 44.44+/-10.20 nM, as well as suppressing HIV-1-induced cytopathic effect with an EC(50) value of 3.04+/-1.20 nM. It also inhibited HIV-2(ROD) and HIV-2(CBL-20) entry and replication in the microM range. Notably, HR212 was highly effective against T20-resistant strains with EC(50) values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/AIDS patients, particularly for those infected by T20-resistant variants.