Prolyl-4-hydroxylase (I) (P4H(I)) is the rate-limiting subunit for the P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we characterized the human P4H(I) promoter for transcription factors and DNA elements regulating P4H(I) expression. Using progressive deletion cloning approach, we constructed pGL3-P4H(I) recombinant plasmids. We identified that a positive regulatory region at positions of -184 to -97bp responsible for approximately 80% of the P4H(I) promoter efficiency. Three E-boxes were located within this region and the E-box at position -135bp explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using ChIP assay. Suppression of USF1 and/or USF2 using specific siRNA resulted in a significant reduction in P4H(I) promoter activity; and overexpressed USF1 or USF2 increased P4H(I) promoter activity significantly. While TGF1 increased the USF1/USF2-E-box binding and P4H(I) promoter activity, this upregulatory effect can be largely prevented by USF1/USF2 specific siRNA. On the other hand, cigarette smoking extracts (CSE), which was shown to suppress P4H(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4H(I) promoter activity. Furthermore, the E-box on the P4H(I) promoter appeared to indiscriminately bind with either USF1 or USF2 with a similar outcome on the promoter efficiency. In conclusion, our study shows that USF1/USF2 plays a critical role in basal P4H(I) expression; both positive (TGF1) and negative regulator (CSE) appears to influence the USF-E-box interaction and affects P4H(I) expression.