Background To immunohistochemically examine the localization of the extracellular matrix (ECM) in the trabecular meshwork (TM) in steroid-induced glaucoma (SG) specimens. Methods We morphologically and immunohistochemically examined six trabecular tissues from three eyes with SG, two with primary open angle glaucoma (POAG), and one without glaucoma. For the morphological study, the ultra-microtome sections were observed using an electron microscope. For the light microscopic immunohistochemical analyses, frozen sections were stained by the avidin-biotin complex method using anti-type IV collagen, anti-type VI collagen, anti-heparan sulfate proteoglycan (HSPG), anti-fibronectin or anti-myocilin (MYOC) antibody. Results The morphological examinations revealed accumulations of basement membrane-like and fine fibrillar-like materials in the outer TM of SG specimen. Type IV collagen, HSPG and fibronectin antibodies in SG specimens showed a greater degree of staining in the outer TM in comparison to the POAG and non-glaucomatous specimens; in contrast, the other antibodies including the type VI collagen and MYOC, did not. Conclusions The localization of ECMs in the TM is different in SG in comparison to that in POAG patients.