We recently reported that XCL1/Lymphotactin has anti-HIV-1 activity and characterized its mechanism of action. Similar to our previous observations with CXCL4/PF4, XCL1 blocks a broad range of HIV-1 isolates, independent of viral coreceptor usage and genetic subtype, and acts by an unconventional mechanism, via direct interaction with HIV-1 gp120. Importantly, HIV-1 inhibition requires access to the alternative, all-beta chemokine conformation of XCL1, which interacts with glycosaminoglycans (GAGs) with high affinity but does not bind/activate the XCL1 receptor. Thus, we examined HIV-1 inhibition by XCL1 after digestion of target cell-surface GAGs with heparitinase. We found that XCL1 was equally effective at blocking HIV-1 in digested and undigested cells, further supporting an antiviral mechanism via direct interaction of XCL1 with HIV-1, rather than binding to GAGs on target cells. Herein, we further investigated the role of GAG binding in XCL1 antiviral activity by testing an XCL1 variant with 2 point mutations (R23A and R43A) that abrogate GAG binding. XCL1 R23A/R43A was unable to block HIV-1 infection across a broad dose range. To validate these data, we showed that XCL1 R23A/R43A could not capture native HIV-1 virions nor block viral attachment/entry. Collectively, these data raise several mechanistic considerations. They suggest that XCL1 requires positively-charged residues (R23/R43) to mediate interaction with negatively-charged domain(s) in gp120. Moreover, the amount, complexity and variability of the glycans that decorate gp120 are likely to influence XCL1 anti-HIV activity. Structure-function studies to delineate key motifs responsible for XCL1-gp120 interactions are underway.