Dimethyladenosine transferase 1 (DIMT1) is an RNA N^6,6-dimethyladenosine (m₂^6,6A) methyltransferase. DIMT1’s role in pre-rRNA processing and ribosome biogenesis is critical for cell proliferation. Here, we investigated the minimal number of residues in a positively charged cleft on DIMT1 required for cell proliferation. We demonstrate that a minimum of four residues in the positively charged cleft must be mutated to alter DIMT1’s RNA-binding ability. The variant (4mutA-DIMT1), which presents reduced RNA binding affinity, is diffuse in the nucleoplasm and nucleolus, in contrast with the primarily nucleolar localization of wild-type DIMT1. The aberrant cellular localization significantly impaired 4mutA-DIMT1’s role in supporting cell proliferation, as shown in competition-based cell proliferation assays. These results identify the minimum region in DIMT1 to target for cell proliferation regulation.