Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1, COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61 bp sequence in FF, ANF, and SF. Knock-down of hc-Krox by siRNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibroblast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1, and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1 and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used, and bind to the COL1A1 promoter in ChIP assays. Moreover, hc-Krox DNA-binding activity to its COL1A1 responsive element is increased in SF, cells producing higher amounts of type I collagen compared to ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery which implicates at least three transcription proteins, hc-Krox, Sp1 and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox.