The cardiac glycoside oleandrin is a main active constituent of the botanical anti-cancer drug candidate PBI-05204, an extract of Nerium oleander. Here, we aimed to determine the circadian sensitivity of mice to oleandrin, and to investigate the role of intestinal P-gp in generating rhythmic drug toxicity. Toxicity and pharmacokinetic experiments were performed with wild-type, Bmal1 (intestine-specific Bmal1 knockout) and Bmal1 (control littermates of Bmal1) mice. The cardiac toxicity (reflected by plasma CK-MB, LDH and cTn-I levels) varied significantly with the times of drug dosing in wild-type mice (a lower toxicity at ZT10 and more severe at ZT2/22). Dosing at ZT2 generated a higher drug exposure than ZT10, supporting a lower toxicity at ZT10. Intracellular accumulation of oleandrin (2.5-10 μM) was reduced in MDCKⅡ-MDR1 than in parental cells. MDR1 overexpression decreased the cell sensitivity to oleandrin toxicity. The net flux ratio (MDCKⅡ-MDR1 versus parental cells) was 2.9 for oleandrin. These data indicated oleandrin as a P-gp substrate. Both mdr1a mRNA and P-gp protein oscillated with the times of the day in small intestine of Bmal1 mice. Intestinal ablation of Bmal1 down-regulated mdr1a mRNA and P-gp protein, and abrogated their rhythms. Likewise, Bmal1 silencing led to down-regulated mdr1a mRNA and to a loss of its rhythmicity in serum-shocked CT26 cells. Based on luciferase reporter assays, Bmal1 regulated rhythmic mdr1a transcription through the clock output genes Hlf and E4bp4. Intestinal ablation of Bmal1 exacerbated oleandrin toxicity and enhanced drug exposure. Moreover, time dependency of toxicity and drug exposure were lost in Bmal1 mice. In conclusion, diurnal intestinal P-gp is a critical factor influencing daily oleandrin exposure and toxicity. Our findings have implications in minimizing oleandrin (and possibly Nerium oleander) toxicity and improving drug efficacy via dosing time optimization.