Rilpivirine is a nonnucleoside reverse transcriptase inhibitor approved for treatment of HIV-1 infection in antiretroviral-naive adult patients. Potential interactions with drug transporters have not been fully investigated. Transport by and inhibition of drug transporters by rilpivirine were analyzed to further understand the mechanisms governing rilpivirine exposure and determine the potential for transporter-mediated drug-drug interactions. The ability of rilpivirine to inhibit or be transported by ABCB1 was determined using ABCB1-overexpressing CEMVBL100 cells and Caco-2 cell monolayers. The Xenopus laevis oocyte heterologous protein expression system was used to clarify if rilpivirine was either transported by or inhibited the function of influx transporters SLCO1A2, SLCO1B1, SLCO1B3, SLC22A2, SLC22A6, and SLC22A8. The ability of rilpivirine to inhibit or be transported by SLC22A1 was determined using SLC22A1-expressing KCL22 cells. Rilpivirine showed higher accumulation in SLC22A1-overexpressing KCL22 cells than control cells (27% increase, P = 0.03) and inhibited the functionality of SLC22A1 and SLC22A2 transport with 50% inhibitory concentrations (IC50s) of 28.5 μM and 5.13 μM, respectively. Inhibition of ABCB1-mediated digoxin transport was determined for rilpivirine, which inhibited digoxin transport in the B-to-A direction with an IC50 of 4.48 μM. The maximum rilpivirine concentration in plasma in patients following a standard 25-mg dosing regimen is around 0.43 μM, lower than that necessary to substantially inhibit ABCB1, SLC22A1, or SLC22A2 in vitro. However, these data indicate that SLC22A1 may contribute to variability in rilpivirine exposure and that interactions of rilpivirine with substrates of SLC22A1, SLC22A2, or ABCB1 may be possible.