We previously showed that numerous polymorphonuclear leukocyte (PMN) granule components efficiently kill Borrelia burgdorferi, the agent of Lyme disease. In addition, motile, granule-poor cytoplasts (U-Cyt) from human blood PMN can exert anti-Borrelia activity against opsonized B. burgdorferi independently of oxidative mechanisms. Here we show that lysates of U-Cyt also possess anti-Borrelia activity, a portion of which comes from the abundant cytosolic protein calprotectin. The anti-Borrelia activity of U-Cyt lysates and recombinant calprotectin was partially or completely reversed by specific antibody to calprotectin and by Zn(2+), a cation essential for the growth of B. burgdorferi and known to inhibit the antimicrobial activity of calprotectin. Quantitative microscopic and regrowth assays revealed that calprotectin acted in a bacteriostatic fashion against B. burgdorferi. We conclude that calprotectin, a potent bacteriostatic agent from a cell primarily recognized for its oxidative and granular antibacterial mechanisms, may play a modulatory role in infection by the Lyme spirochete, particularly at sites of acute inflammation.