Bioluminescence (BL) imaging based on D-luciferin (D-luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the D-luc–luciferase reaction, an approach to increase intracellular levels of D-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a D-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of D-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of D-luc was higher than that in control cells, and the specific D-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 μM. The interaction between OAT1 and D-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that D-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent Km of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of D-luc could be a rate-limiting step in the D-luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of D-luc. Our findings suggest that because OAT1 is capable of transporting D-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.