Retrocyclin (RC)-101 is a cationic theta-defensin that inhibits HIV-1 entry. Passaging HIV-1(BAL) under selective pressure by this cyclic minidefensin resulted in only a 5- to 10-fold decrease in viral susceptibility to RC-101. Emergent viral isolates had three amino acid substitutions in their envelope glycoprotein. One was in a CD4-binding region of gp120, and the others were in the heptad repeat (HR) domains of gp41 (HR1 and HR2). Each mutation replaced an electroneutral or electronegative residue with one that was positively charged. These mutations were evaluated either alone or in combination in a single-round viral entry assay. Although the mutation in gp120 did not affect viral entry, the mutation in HR1 of gp41 conferred relative resistance to RC-101. Interestingly, the envelope with the HR2 mutation was less efficient and became codependent on the presence of RC-101 for entry. The adaptive response of HIV-1 to this cationic host defense peptide resembles the responses of bacteria that modulate their surface or membrane charge to evade analogous host defense peptides. These findings also suggest that interactions between theta-defensins and gp41 may contribute to the ability of these cyclic minidefensins to prevent HIV-1 entry into target cells.