We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s 4 dU) 35 ]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s 4 dU) 35 is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC 50 : 0.8–25.4 μg/ml) in vitro. Besides its previously reported anti-RT activity, (s 4 dU) 35 mediated its antiviral action by preventing virus attachment (IC 50 : 0.002–0.003 μg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s 4 dU) 35 preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s 4 dU) 35 did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s 4 dU) 35 as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.