Neurological side effects consistent with ivermectin toxicity have been observed in dogs when high doses of the common heartworm prevention agent ivermectin are coadministered with spinosad, an oral flea prevention agent. Based on numerous reports implicating the role of the ATP-binding cassette drug transporter P-glycoprotein (P-gp) in ivermectin efflux in dogs, an in vivo study was conducted to determine whether ivermectin toxicity results from a pharmacokinetic interaction with spinosad. Beagle dogs were randomized to three groups treated orally in parallel: Treatment group 1 (T01) received ivermectin (60 μg/kg), treatment group 2 (T02) received spinosad (30 mg/kg), and treatment group 3 (T03) received both ivermectin and spinosad. Whereas spinosad pharmacokinetics were unchanged in the presence of ivermectin, ivermectin plasma pharmacokinetics revealed a statistically significant increase in the area under the curve (3.6-fold over the control) when ivermectin was coadministered with spinosad. The majority of the interaction is proposed to result from inhibition of intestinal and/or hepatic P-gp-mediated secretory pathways of ivermectin. Furthermore, in vitro Transwell experiments with a human multidrug resistance 1-transfected Madin-Darby canine kidney II cell line showed polarized efflux at concentrations ≤2 μM, indicating that spinosad is a high-affinity substrate of P-gp. In addition, spinosad was a strong inhibitor of the P-gp transport of digoxin, calcein acetoxymethyl ester (IC 50 = 3.2 μM), and ivermectin (IC50 = 2.3 μM). The findings suggest that spinosad, acting as a P-gp inhibitor, increases the risk of ivermectin neurotoxicity by inhibiting secretion of ivermectin to increase systemic drug levels and by inhibiting P-gp at the blood-brain barrier.