The actinomycete-derived lectin actinohivin (AH) inhibits entry of HIV-1 to susceptible cells at low nM concentrations. The cooperative binding of three segments of AH to three high mannose-type glycans (HMTGs) of HIV-1 gp120 generates specific and strong anti-HIV activity. Dimerization of AH effectively improves anti-HIV activity by increasing the number of HMTG-binding pockets. AH dimers were prepared using an Escherichia coli expression system and their anti-syncytium formation and anti-HIV activities were evaluated. Each dimer was constructed by a head-to-tail fusion of two AH molecules, with or without a spacer. As a result, His-TEV-AH/RTB(132-143)/AH, which has the residues 132-143 of ricin toxin B-chain (RTB) as a spacer, had 20-fold higher anti-syncytium formation activity and also exhibited 2-30-fold higher anti-HIV activity than AH against various clinically isolated HIV-1 strains, including drug-resistant ones. Mutation analysis implies that all six HMTG-binding pockets of the dimer participated in HMTG binding. Several AH dimers with different spacer sequences showed diverse activities, suggesting that the spacer sequence is an important factor to create higher anti-HIV activity. A dimer with improved anti-HIV activity would be a good candidate for investigation as a potential microbicide to prevent HIV transmission.