Transporters are major determinants of the disposition of xenobiotics and endogenous chemicals in the body. Organic anion transporter 3 (Oat3) functions in the kidney and brain to remove metabolic waste, toxins, and drugs, and thus transports diverse chemicals. Some β-lactam antibiotics interact with Oat3, and penicillin G exhibits a strong dependence on Oat3 for renal elimination. However, over 80 β-lactams exist, and many have not been assessed for an interaction with Oat3. Moreover, β-lactams continue to receive U.S. Food and Drug Administration approval. This study identified new β-lactam-Oat3 interactions, provided a head-to-head comparison with Oat1, and characterized the physicochemical determinants of affinity for Oat3. Cells expressing mouse Oat3 (mOat3) and Oat1 (mOat1), and human OAT3 (hOAT3) were used to test inhibitors, and high-performance liquid chromatography (HPLC) was used to measure transport. Of 26 β-lactams tested, 12 were clear inhibitors of Oat3, and 14 exhibited poor interactions. Inhibitors exhibited a nearly identical rank-order of potency against mOat3 and hOAT3. Oat1 demonstrated a poor interaction with most β-lactams. The majority of Oat3 inhibitors were substrates, and there were clear physicochemical differences between inhibitors and noninhibitors. That is, inhibitors had nearly 40% fewer hydrogen bond donors (P < 0.001), a lower total polar surface area (P < 0.05), and greater lipophilicity (LogP of inhibitors, +1.41; noninhibitors, -1.54; P < 0.001). Pharmacophore mapping revealed a prohibitive hydrogen bond donor group in noninhibitors adjacent to a hydrophobic moiety that was important for binding to Oat3. These findings indicate that Oat3 recognizes lipophilic β-lactams more readily. Moreover, this study has potential implications for designing β-lactams to avoid renal accumulation or brain efflux via Oat3.