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ZNF16 (also known as HZF1 and KOX9) is a multi-C2H2 zinc finger protein first identified via its expression in human T-cells and shown to have a role in blood cell differentiation. ZNF16 was later shown to be ubiquitously expressed in a variety of fetal and adult tissues, suggesting a broader function. In this study, we confirm the ubiquitous expression of ZNF16 in a variety of cancer and non-cancer cell lines and show that ZNF16 depletion reduces cell viability in all cell lines tested. Furthermore, we show that ZNF16 localizes to the nucleolus in a transcription-dependent manner, interacts with the intergenic spacer region of the rDNA and promotes rDNA transcription. Additionally, RNA-seq experiments after ZNF16 depletion revealed that ZNF16 also has roles in a variety of pathways including ECM-receptor interaction, focal adhesions, cytokine-cytokine receptor interactions, human papillomavirus (HPV) infection and cancer pathways. These findings are consistent with broader roles for ZNF16, including the regulation of nucleolar function, a process that is essential for all cells, and provide evidence at the cellular/molecular level of its role in the regulation of cancer-associated genes (e.g., NRAS, BIRC3, EGFR).