Rax1 and Rax2 proteins provide the spatial landmark signal during the bipolar budding of Saccharomyces cerevisiae for the proper assembly of the new bud. The nonconventional yeast Yarrowia lipolytica also undergoes bipolar budding, and its genome encodes YlRax1 (YALI0E10329) and YlRax2 (YALI0A04609), the orthologs of Rax1 and Rax2, respectively. In this study, we explored the roles of YlRax1 and YlRax2 in the bipolar budding of Y. lipolytica. Deletion of YlRax1 and YlRax2 caused a proportion of Y. lipolytica cells to exhibit unipolar budding. Furthermore, our results revealed that YlRax1 and YlRax2 were localized at the mother-bud neck as well as the previous division sites, and their localization to the division sites was persistent through multiple cell cycles. Moreover, our study demonstrated that the 100 amino acids at the N-terminal of YlRax1 are not essential for its function. In contrast, the transmembrane domains at the C-terminal of YlRax1 and YlRax2 are essential for their normal function, as the truncated protein fragments with deleted TM domains could not restore the normal functioning of the corresponding strains with knocked-out YlRax1 or YlRax2. These results indicate that YlRax1 and YlRax2 are involved in partially regulating bipolar budding and that these two proteins are interdependent in localization and function. Furthermore, our results indicate that there will be novel landmark proteins regulating its bipolar budding in Y. lipolytica.