Insulin-like growth factor binding protein-1 (IGFBP-1) can bind to the alpha5beta1 integrin and stimulate cellular migration in Chinese hamster ovary (CHO) cells. IGFBP-1 is a major product of the endometrium of pregnancy (decidua), and may interact with invading cytotrophoblasts expressing alpha5beta1 integrin to modulate their invasion. The present study investigated IGFBP-1 interaction with cytotrophoblast alpha5beta1 integrin, and its effects on trophoblast attachment to fibronectin and invasion into decidualized endometrial stromal cell multilayers. IGFBP-1 incubated with cytotrophoblast extracts was co-precipitated by an antibody to the alpha5 integrin subunit. Up to 55% of radiolabeled IGFBP-1 bound to cytotrophoblasts was displaced by excess non-radioactive IGFBP-1, but not by IGFBP-3. Cytotrophoblast attachment to fibronectin was inhibited by an RGD-containing octapeptide, by antibodies to the alpha5 subunit or the alpha5beta1 heterodimer, and by IGFBP-1. Cytotrophoblasts showed limited invasion into endometrial stromal multilayers decidualized in vitro secreting abundant IGFBP-1, but invaded multilayers when IGFBP-1 production was inhibited by insulin. Invasion into insulin-treated multilayers was prevented by addition of exogenous IGFBP-1 but not by IGFBP-3. These findings suggest IGFBP-1 may modulate trophoblast invasiveness.