Lyme disease (LD) is caused by the spirochete Borrelia burgdorferi sensu lato (Bbsl). After transmission to humans by ticks, Bbsl spreads to multiple organs, leading to arthritis, carditis, and neuroborreliosis. No effective prophylaxis against human LD prior to tick exposure is currently availa-ble. Thus, a pre-exposure prophylaxis (PrEP) against LD is needed. The establishment of LD bacteria at diverse sites is dictated partly by the binding of Bbsl to proteoglycans (PGs) and glycosaminoglycans (GAGs) in tissues. The drug heparin is structurally similar to these GAGs and inhibits Bbsl attach-ment to PGs, GAGs, cells, and tissues, suggesting its potential to prevent LD. However, the anticoagulant activity of heparin often results in hemorrhage, hampering the development of this compound as LD PrEP. We have previously synthesized a non-anticoagulant version of heparins (NACHs), which was verified for safety in mice and humans. Here, we showed that NACH blocks Bbsl attachment to PGs, GAGs, and mammalian cells. We also found that treating mice with NACH prior to the exposure of ticks carrying Bbsl followed by continuous admin-istration of this compound prevents tissue colonization by Bbsl. Furthermore, NACH-treated mice develop greater levels of IgG and IgM against Bbsl at early stages of infection, sug-gesting that the upregulation of antibody immune responses may be one of the mechanisms for NACH-mediated LD pre-vention. This is one of the first studies examining the ability of a heparin-based compound to prevent LD prior to tick ex-posure. The information presented might also be extended to prevent other infectious diseases agents.