Insulin-like growth factor (IGF) and epidermal growth factor (EGF) regulate various physiological and pathological processes. The IGF binding protein (IGFBP)-3 regulates cell proliferation in IGF-dependent and independent fashions. Recently, we identified IGFBP-3 as a novel EGFR downstream target molecule in primary and immortalized human esophageal epithelial cells, suggesting interplay between the EGF and IGF-signaling pathways. However, the regulatory mechanisms for IGFBP-3 expression and its functional role in esophageal cell proliferation remain to be elucidated. Herein, we report that IGFBP-3 mRNA and protein were induced upon growth factor deprivation in primary and immortalized human esophageal cells through mechanisms requiring p53-independent de novo mRNA transcription and protein synthesis. This occurred in the face of the activated phosphatidylinositol 3-OH-kinase (PI3K)- mammalian target of rapamycin (mTOR) pathway. Secreted IGFBP-3 neutralized IGFs and prevented IGF-IR activation. By contrast, EGF suppressed IGFBP-3 mRNA and protein expression through activation of mitogen activated protein kinase (MAPK) in an EGFR-tyrosine kinase-dependent manner to restore the cellular response to IGF-I. When stably overexpressed, wild-type IGFBP-3, but not I56G/L80G/L81G (GGG) mutant IGFBP-3 that has a reduced affinity to IGFs, prevented IGF-I from activating IGF-IR and AKT as well as stimulating cell proliferation. However, unlike other cell types where IGFBP-3 exerts antiproliferative effects, neither wild-type nor GGG mutant IGFBP-3 alone affected cell proliferation, or EGFR activity. These results indicate that IGF-signaling is subject to negative regulation through IGFBP-3 and positive regulation by EGF, the latter of which suppresses IGFBP-3. This provides a platform for understanding a novel crosstalk between EGF and IGF mediated pathways.