Yarrowia lipolyticais a dimorphic fungus used as a model organism to investigate diverse biotechnological and biological processes, such as cell differentiation, heterologous protein production, and bioremediation strategies. However, little is known about the biological processes responsible for cation concentration homeostasis. Metals play pivotal roles in critical biochemical processes, and some are toxic at unbalanced intracellular concentrations. Membrane transport proteins control intracellular cation concentrations. Analysis of theY. lipolyticagenome revealed a characteristic functional domain of the cation efflux protein family, i.e., YALI0F19734g, which encodes YALI0F19734p (a putativeYl-Dmct protein), which is related to divalent metal cation tolerance. We report the in silico analysis of the putativeYl-Dmct protein’s characteristics and the phenotypic response to divalent cations (Ca2+, Cu2+, Fe2+, and Zn2+) in the presence of mutant strains, Δdmctand Rdmct, constructed by deletion and reinsertion of theDMCTgene, respectively. The absence of theYl-Dmct protein induces cellular and growth rate changes, as well as dimorphism differences, when calcium, copper, iron, and zinc are added to the cultured medium. Interestingly, the parental and mutant strains were able to internalize the ions. Our results suggest that the protein encoded by theDMCTgene is involved in cell development and cation homeostasis inY. lipolytica.