A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.