Annexin B1 from Cysticercus cellulosae has recently been identified using immunological screening in an attempt to find novel antigens for vaccine development against cysticercosis. The protein possesses anticoagulant activity and carries significant therapeutic potential due to its thrombus-targeting and thrombolytic properties. We investigated the biochemical properties of annexin B1 using liposome and heparin Sepharose copelleting assays, as well as CD spectroscopy. The calcium-dependent binding to acidic phospholipid membranes is reminiscent of other mammalian annexins with a clear preference for high phosphatidylserine content. A unique property of annexin B1 is its ability to bind to liposomes with high phosphatidylserine content in the absence of calcium, which might be due to the presence of several basic residues on the convex protein surface that harbours the membrane-binding loops. Annexin B1 demonstrates lectin properties and binds to heparin Sepharose in a cooperative, calcium-dependent manner. Although this binding is reversible to a large extent, a small fraction of the protein remains bound to the glycosaminoglycan even in the presence of high concentrations of EDTA. Analogous to annexin A5, we propose a model of heparin wrapped around the protein thereby engaging in calcium-dependent and calcium-independent interactions. Although the calcium-independent heparin-binding sites identified in annexin A5 are not conserved, we hypothesize three possible sites in annexin B1. Results from CD spectroscopy and thermal denaturation indicate that, in solution, the protein binds calcium with a low affinity that leads to a slight increase in folding stability.