Laminin-α2 chain is one of the major constituent proteins of the basement membrane in the mammalian testis. The laminin-type globular (LG) domains of LG3, 4 and 5 (LG3/4/5, an 80 kDa fragment) can be cleaved from laminin-α2 chain at the C-terminus via the action of matrix metalloproteinase 9 (MMP-9). This LG3/4/5 is a biologically active fragment, capable of modulating the Sertoli cell blood–testis barrier (BTB) function by tightening the barrier both in vitro and in vivo. Overexpression of LG3/4/5 cloned into a mammalian expression vector pCI-neo in Sertoli cells in a Sertoli cell in vitro model with a functional BTB also protected Sertoli cells from cadmium chloride (CdCl2, an environmental toxicant) mediated cell injury. Importantly, overexpression of LG3/4/5 in the testis in vivo was found to block or rescue cadmium-induced BTB disruption and testis injury. LG3/4/5 was found to exert its BTB and spermatogenesis promoting effects through corrective spatiotemporal expression of actin- and MT-based regulatory proteins by maintaining the cytoskeletons in the testis, illustrating the therapeutic implication of this novel bioactive fragment.