Transforming growth factor beta 1-induced (TGFBI) protein is an extracellular matrix (ECM) protein that is associated with other ECM proteins and functions as a ligand for various types of integrins. In this study, we investigated how human TGFBI expression is regulated in lung and breast cancer cells. We observed that the TGFBI promoter in A549 and MBA-MD-231 cells, which constitutively express TGFBI, existed in an open chromatin conformation associated with transcriptionally permissive histone modifications. Moreover, we found that TGFBI expression required Sp1 transcription elements that can bind transcription factors Sp1 and Sp3 in vitro. Occupancy of the TGFBI promoter by Sp1 and Sp3 in vivo was only observed in TGFBI-expressing cells, indicating that open chromatin conformation might facilitate the binding of Sp1 and Sp3 to the TGFBI promoter region. TGFBI promoter activity was impaired when Sp1 elements were mutated, but was increased when Sp1 or Sp3 factors was overexpressed. Furthermore, Sp1 inhibition in vivo by mithramycin A, as well as knockdown of Sp1 and/or Sp3 expression by short interfering RNA, significantly reduced TGFBI mRNA and protein levels. Thus, our data demonstrated that the expression of TGFBI is well correlated with chromatin conformation at the TGFBI promoter, and that factors Sp1 and Sp3 are the primary determinants for the control of constitutive expression of TGFBI gene.