The human cytomegalovirus (HCMV) UL138 protein downregulates the cell surface expression of the multidrug resistance-associated protein 1 (MRP1) transporter. We examined the genetic requirements within UL138 for MRP1 downregulation. We determined that the acidic cluster dileucine motif is essential for UL138-mediated downregulation of MRP1 steady-state levels and inhibition of MRP1 efflux activity. We also discovered that the naturally occurring UL138 protein isoforms, the full-length long isoform of UL138 and a short isoform missing the N-terminal membrane-spanning domain, have different abilities to inhibit MRP1 function. Cells expressing the long isoform of UL138 show decreased MRP1 steady-state levels and fail to efflux an MRP1 substrate. Cells expressing the short isoform of UL138 also show decreased MRP1 levels, but the magnitude of the decrease is not the same, and they continue to efficiently efflux an MRP1 substrate. Thus, the membrane-spanning domain, while dispensable for a UL138-mediated decrease in MRP1 protein levels, is necessary for a functional inhibition of MRP1 activity. Our work defines the genetic requirements for UL138-mediated MRP1 downregulation and anticipates the possible evolution of viral escape mutants during the use of therapies targeting this function of UL138.IMPORTANCE HCMV UL138 curtails the activity of the MRP1 drug transporter by reducing its steady-state levels, leaving cells susceptible to killing by cytotoxic agents normally exported by MRP1. It has been suggested in the literature that capitalizing on this UL138-induced vulnerability could be a potential antiviral strategy against virally infected cells, particularly those harboring a latent infection during which UL138 is one of the few viral proteins expressed. Therefore, identifying the regions of UL138 required for MRP1 downregulation and predicting genetic variants that may be selected upon UL138-targeted chemotherapy are important ventures. Here we present the first structure-function examination of UL138 activity and determine that its transmembrane domain and acidic cluster dileucine Golgi sorting motif are required for functional MRP1 downregulation.