CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a component of the euchromatic Setd1A histone H3K4 methyltransferase complex, and is a critical regulator of histone methylation, cytosine methylation, cellular differentiation, and vertebrate development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but show increased levels of global histone H3K4 methylation, suggesting that Cfp1 functions to inhibit or restrict the activity of the Setd1A histone H3K4 methyltransferase complex. The studies reported here reveal that ES cells lacking Cfp1 contain decreased levels of Setd1A and show subnuclear mislocalization of both Setd1A and trimethylation of histone H3K4 with regions of heterochromatin. Remarkably, structure-function studies reveal that expression of either the N-terminal fragment of Cfp1 (amino acids 1-367) or the C-terminal fragment of Cfp1 (amino acids 361-656) is sufficient to restore appropriate levels of Setd1A in CXXC1(-/-) ES cells. Furthermore, functional analysis of various Cfp1 point mutations reveals that retention of either Cfp1 DNA-binding activity or association with the Setd1 histone H3K4 methyltransferase complex is required to restore normal Setd1A levels. In contrast, expression of full-length Cfp1 in CXXC1(-/-) ES cells is required to restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicating that both Cfp1 DNA-binding activity and interaction with the Setd1A complex are required for appropriate genomic targeting of the Setd1A complex. These studies illustrate the complexity of Cfp1 function, and identify Cfp1 as a regulator of Setd1A genomic targeting.