UHRF1 facilitates the establishment and maintenance of DNA methylation patterns in mammalian cells. The establishment domains are defined, including E3 ligase function, but the maintenance domains are poorly characterized. Here, we demonstrate that UHRF1 histone- and hemimethylated DNA binding functions, but not E3 ligase activity, maintain cancer-specific DNA methylation in human colorectal cancer (CRC) cells. Disrupting either chromatin reader activity reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes (TSGs), and reduces CRC oncogenic properties. Moreover, an inverse correlation between high UHRF1 and low TSG expression tracks with CRC progression and reduced patient survival. Defining critical UHRF1 domain functions and its relationship with CRC prognosis suggests directions for, and value of, targeting this protein to develop therapeutic DNA demethylating agents. Display omitted •UHRF1 maintains cancer-specific DNA methylation through its chromatin reader domains•PHD and SRA domain mutants phenocopy UHRF1 depletion to reverse DNA hypermethylation•Disrupting PHD or SRA domain functions impairs key oncogenic properties of CRC cells•The maintenance function of overexpressed UHRF1 in CRC has prognostic significance Kong et al. show that histone and hemimethylated DNA reader domains are critical for UHRF1 to maintain aberrant DNA methylation in colorectal cancer (CRC) cells. Blocking either domain reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes, and reduces CRC oncogenic properties.