Near the base of mammalian seminiferous epithelium, Sertoli cells are joined by tight junctions, which constitute the blood–testis barrier. Differentiating germ cells are completely enveloped by Sertoli cells and must traverse the tight junctions during spermatogenic cycle. Following the specific ligand activation of L-selectin, the up-regulated Rho family small G-proteins have been implicated as important modulators of tight junctional dynamics. Although the activation of L-selectin transmits subsequent intracellular signals in a Ca+2-dependent fashion in various cell types, little is understood regarding the signaling pathways utilized by L-selectin in Sertoli cells. Therefore, we have examined the possible resultant calcium influx triggered by specific ligand-activation of cell surface L-selectin receptors or by cross-linking of L-selectin with anti-L-selectin. Spectrofluorimetric studies demonstrate increase of intracellular Ca+2 levels immediately after the treatment of the L-selectin ligands, fucoidan and sialyl Lewis-a, or after treatment with anti-L-selectin antibody. We then determined the mechanism of Ca+2 influx by investigating L- and T-type voltage-operated Ca+2 channels, which have been suggested to present in the membranes of Sertoli cells. Data demonstrate that Sertoli cells treated with L-type voltage-operated Ca+2 channel antagonists, nifedipine, diltiazem, or verapamil, lead to dose-dependent blockage of L-selectin-induced Ca+2 influx. Cells treated with mibedradil, a T-type voltage-operated Ca+2 channel antagonist, results in little or no blocking effect. Therefore, we conclude that activation of Sertoli cell L-selectin induces Ca+2 influx, which is at least partially regulated by L-type voltage-operated Ca+2 channels. J. Cell. Biochem. 101: 1023–1037, 2007. © 2007 Wiley-Liss, Inc.