P-glycoprotein (P-gp) largely influences the pharmacokinetics (PK) and toxicities of xenobiotics in a patient-specific manner so that personalized drug scheduling may lead to significant patient's benefit. This systems pharmacology study investigated P-gp activity in mice according to organ, sex, feeding status, and circadian time. Sex-specific circadian changes were found in P-gp ileum mRNA and protein levels, circadian amplitudes being larger in females as compared to males. Plasma, ileum and liver concentrations of talinolol, a pure P-gp substrate, significantly differed according to sex, feeding and circadian timing. A physiologically-based PK model was designed to recapitulate these datasets. Estimated mesors (rhythm-adjusted mean) of ileum and hepatic P-gp activity were higher in males as compared to females. Circadian amplitudes were consistently higher in females and circadian maxima varied by up to 10 h with respect to sex. Fasting increased P-gp activity mesor and dampened its rhythm. Ex-vivo bioluminescence recordings of ileum mucosae from transgenic mice revealed endogenous circadian rhythms of P-gp protein expression with a shorter period, larger amplitude, and phase delay in females as compared to males. Importantly, this study provided model structure and parameter estimates to refine PK models of any P-gp substrate to account for sex, feeding and circadian rhythms.