The alkaline phosphatase (ALP) gene is an important marker of osteoblast differentiation and bone formation. Although the molecular mechanisms of increased ALP expression in response to all-trans retinoic acid (ATRA) have been reported, the role of ATRA in chromatin structure changes remains unknown. Our results show that the expression of mouse liver, bone, and kidney ALP (mL/B/K-ALP) induced by ATRA in C3H10T 1/2 cells was related to the retinoic acid nuclear receptors, RARα and RARβ, which are not involved in the MAPK pathway. DNase I hypersensitivity analysis revealed an inducible hypersensitive site in the mL/B/K-ALP promoter at ~520 bp upstream of the transcription start site. Chromatin immunoprecipitation experiments showed a cascade of transcription cofactor recruitment events during ATRA-induced upregulation of mL/B/K-ALP. Together, our results provide a link between ATRA-induced mL/B/K-ALP gene transcription and chromatin remodeling.