The expression of P-glycoprotein (P-gp), an ATP-dependent efflux transporter, is closely associated with the failure of chemotherapy and drug absorption. Two synthesized optically active phenylbutenoid dimers, 3S-(3,4-dimethoxyphenyl)-4R-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (1) and 3R-(3,4-dimethoxyphenyl)-4S-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (2), were tested for their P-gp inhibitory effects by measuring cellular accumulation and efflux of daunomycin in P-gp-overexpressed human breast cancer cells (MCF-7/ADR). Compound 2 significantly increased the accumulation of daunomycin (539%) and decreased the efflux of this compound (55.4%), and similar results were observed for 1. ATPase assays and Western blot analysis were performed to identify the mechanisms by which compounds 1 and 2 inhibit P-gp. In addition, changes in the pharmacokinetic profile of paclitaxel coadministered with 2 in rats were evaluated. Paclitaxel (25 mg/kg) when orally administered with 2 (5 mg/kg) improved its relative bioavailability by 185%. Compound 2 effectively improved cellular accumulation by reducing the efflux of daunomycin and significantly enhanced oral exposure to paclitaxel. Therefore, compound 2 may be useful for improving oral exposure and cellular availability of drugs that are also substrates of P-gp.