Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial inter-patient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b-/-) mice, hydroxyurea PK was analyzed in vivo by measuring 14C-hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled ¹⁴CO₂ metabolite. Plasma levels were significantly reduced by 20% in Oatp1b-/- mice compared to wild-type (AUC of 38.64 μg-hr/ml or 48.45 μg-hr/ml, respectively) after oral administration; whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by 2-fold in Oatp1b-/- mice (356.9 pmol/g vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. , Hydroxyurea accumulation in the liver was also decreased (136.6 pmol/g vs. 107.3 pmol/g in wild-type or Oatp1b-/- mice, respectively) correlating with a decrease in exhaled ¹⁴CO₂. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.