Pain in sickle cell anemia (SCA) is clinically managed with opioid analgesics. There are reports that SCA patients tolerate high doses of these drugs without adequate pain relief. The current study investigated the in vitro hepatic metabolism of opioids in mouse models of sickle cell anemia, with the hypothesis that higher dose requirements in SCA could be explained by an increased metabolism rate of opioids. Various rodent cytochrome P450 substrates, i.e., buprenorphine and codeine, and rodent uridine glucuronosyl-transferase substrates, i.e., morphine, buprenorphine, and estradiol, were studied. The three groups used were: 1) control C57BL mice, 2) mice with the human α-globin and sickle β-globin transgenes (SC), and 3) mice with the human α-globin and sickle β-globin transgenes, and homozygous for the murine α-globin and heterozygous for the βmajor-gene knockout (SCKO). In vitro hepatic microsomal incubations were carried out for each substrate, and data were fit to the Michaelis-Menten equation. Morphine formation had a higher Vmax in SCKO microsomes (0.4 ± 0.009 nmol/min · mg; estimate ± S.E.) than controls (0.25 ± 0.007). Morphine-3-glucuronide formation had Vmax estimates of 18.9 ± 0.6, 25.1 ± 0.4, and 27.06 ± 1.1 nmol/min · mg in control, SC, and SCKO microsomes, respectively. The control Vmax for estradiol-3-glucuronide formation was 2-fold greater than in SCKO microsomes. The control Vmax for estradiol 17-glucuronide formation was 3.4- and 2.2-fold greater than in SC and SCKO microsomes. Thus, in vitro metabolism of opioids is altered in SCA mouse models, which may lead to altered clearances of these drugs.