In mammals it is unclear if UHRF1-mediated DNA maintenance methylation by DNMT1 is strictly dependent on histone H3K9 methylation. Here we have generated an Uhrf1 knockin (KI) mouse model that specifically abolishes the H3K9me2/3-binding activity of Uhrf1. The homozygous Uhrf1 KI mice are viable and fertile, and exhibit ∼10% reduction of DNA methylation in various tissues. The reduced DNA methylation occurs globally in the genome and does not restrict only to the H3K9me2/3 enriched repetitive sequences. In vitro UHRF1 binds with higher affinity to reconstituted nucleosome with hemi-methylated CpGs than that with H3K9me2/3, although it binds cooperatively to nucleosome with both modifications. We also show that the nucleosome positioning affects the binding of methylated DNA by UHRF1. Thus, while our study supports a role for H3K9 methylation in promoting DNA methylation, it demonstrates for the first time that DNA maintenance methylation in mammals is largely independent of H3K9 methylation.