Prolyl-4-hydroxylase alpha(I) (P4Halpha(I)) is the rate-limiting subunit for P4H enzyme activity, which is essential for procollagen hydroxylation and secretion. In the current study, we have characterized the human P4Halpha(I) promoter for transcription factors and DNA elements regulating P4Halpha(I) expression. Using a progressive deletion cloning approach, we have constructed pGL3-P4Halpha(I) recombinant plasmids. We have identified a positive regulatory region at the positions of bp -184 to -97 responsible for approximately 80% of the P4Halpha(I) promoter efficiency. Three E-boxes were located within this region, and the E-box at position bp -135 explains most of the regulatory capacity. Upstream stimulatory factors (USF1/USF2) were shown to bind on the E-box using chromatin immunoprecipitation assay. Suppression of USF1 and/or USF2 using specific short interference RNA resulted in a significant reduction in P4Halpha(I) promoter activity, and overexpressed USF1 or USF2 increased P4Halpha(I) promoter activity significantly. Although transforming growth factor beta1 increased the USF1/USF2-E-box binding and P4Halpha(I) promoter activity, this up-regulatory effect can be largely prevented by USF1/USF2-specific short interference RNA. On the other hand, cigarette smoking extracts, which have been shown to suppress P4Halpha(I) expression, inhibited the binding between the USF1/USF2 and E-box, resulting in a reduced P4Halpha(I) promoter activity. Furthermore, the E-box on the P4Halpha(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 P4Halpha(I) expression, and both positive (transforming growth factor beta1) and negative (cigarette smoking extract) regulators appear to influence the USF-E-box interaction and affect P4Halpha(I) expression.