Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with complex and diverse phenotypes, including overgrowth. BWS is caused by aberrant hypermethylation of imprinting control region within IGF2-H19 locus (H19-ICR) on the maternal allele, which is normally unmethylated. Although mutations in the binding site of CTCF or OCT4 on the maternal H19-ICR cause aberrant hypermethylation and result in BWS, its molecular mechanism is not fully understood. In particular, the responsible sequence(s) for aberrant hypermethylation inducing BWS-like overgrowth, has not been identified in mice. To address this, we generated 10 strains of mutant mice harboring SOX2-OCT4 binding site (SOBS) and/or four CTCF binding sites (CTS1-4) in H19-ICR, either solely or simultaneously. We found that double mutations of SOBS/CTS3 or SOBS/CTS4 showed aberrant hypermethylation of the entire H19-ICR, biallelic expression of Igf2, decreased expression of H19, and overgrowth. On the other hand, CTS3/CTS4 double mutant mice showed limited hypermethylation to the regions encompassing CTS3, CTS4 and SOBS, and exhibited less frequent overgrowth compared to the SOBS/CTS3 mutants. Other mutant mice did not show significant changes in DNA methylation except at the mutated site(s), and their body weights were in normal range. Additionally, the accumulation of CTCF and RAD21 on CTS1-4 was significantly reduced in SOBS/CTS3 mutants, but did not change in SOBS/CTS4 or CTS3/CTS4 mice. These findings suggest that SOBS and CTS3 are essential for maintaining the unmethylated state of the maternal H19-ICR in mice.