Arrest-defect-1 protein (ARD1), an acetyltransferase, catalyzes N—α-acetylation in yeast. In mammalian cells, both N-α-acetylation and ε-acetylation induced by ARD1 have been reported. Emerging evidence has revealed that ARD1 is involved in a variety of cellular functions, including proliferation, apoptosis, autophagy, and differentiation and that dysregulation of ARD1 is associated with tumorigenesis and neurodegenerative disorder. This review will discuss recent discoveries regarding variations among the different ARD1 isoforms, the associated biological functions of ARD1, and ARD1 localization in different cells. We will also discuss the potential upstream regulators and downstream targets of ARD1 to provide new avenues for resolving its controversial roles in cancer development.