Polyethylene glycol 400 (PEG400) is a commonly used co-solvent. Recent studies suggested that it might affect the systemic exposure of oral drugs based on mechanisms besides increasing their solubility. In this study, we systematically explored the dose related effects and underlying mechanisms of PEG400 on the systemic exposure of oral berberine, a new drug candidate having multiple pharmacological effects. The results showed that low concentration (5% and 15%) PEG400 decreased but high concentration (50%) PEG400 increased the absorption of berberine across mouse gut sacs by 6.5-fold. As expected, 50% PEG400 increased the solubility of berberine from 1.7 to 10.5 mg mL⁻¹. Surprisingly, 50% PEG400 reversibly opened intestinal paracellular tight junctions, which was verified by the increased absorption of the marker compound, fluorescein isothiocyanate-labeled dextran (molecular weight [M_w]: 4400 g mol⁻¹, FD4). However, 50% PEG400 did not influence the intestinal efflux, liver uptake, or hepatic metabolism of berberine. The systemic exposure (AUC_{0–4 h}) of berberine in mice receiving orally administered berberine (200 mg kg⁻¹) dissolved in water or 50% PEG400 reached 30.6 ng h mL⁻¹ or 188.1 ng h mL⁻¹, respectively. The enhancive effect of PEG400 was significantly inhibited by cycloheximide pretreatment. In conclusion, our data showed that high concentration of PEG400 increased the systemic exposure of berberine by increasing its solubility, prompting its intestinal paracellular absorption and lymphatic transport. Our results provide new insights into the effects and mechanisms of PEG400 on the systemic exposure of oral drugs. In addition, the results would encourage further exploration on the application of PEG400 in drug delivery.