Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5' non-coding region of the chicken alpha-globin domain. Here, we demonstrate that this element, called the alpha EHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult alpha-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the alpha EHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the alpha EHS-1.4 insulator contributes in organizing the chromatin structure of the alpha-globin gene domain and prevents activation of adult alpha-globin gene expression at the erythroblast stage via CTCF.