This study aimed to elucidate the clinical efficacy and pharmacokinetics of levothyroxine (LT4) suppository, thus, we examined the pharmacokinetics of thyroxine (T4) after the administration of the suppository in thyroidectomized rats and examined dose and the levels of free T4 (FT4) in patients with hypothyroidism receiving suppositories. Thyroidectomized rats were administered with LT4 solution and LT4 suppository (30 µg/kg), and plasma T4 concentrations were measured using LC/MS. The AUC _0–168 of T4 after rectal administration of the LT4 suppository was 64% lower than these values after oral administration. To evaluate clinical effect of LT4 suppository, we enrolled 6 Japanese patients with hypothyroidism (2 men and 4 women; age, 68.2±13.5 years) who were administered LT4 suppository at Kameda Medical Center from 2007 to 2013 in this case series. The FT4 level during the administration of suppositories was significantly lower than that during the administration of tablets (0.657±0.183 ng/dL vs. 1.25±0.51 ng/dL, p =0.034). The FT4/dose ratio for the suppository was significantly 44% lower than that for the tablet ( p =0.020). In conclusion, although the bioavailability of LT4 is lower after administration of the suppository than after the oral formulation, it was suggested that T4 levels can be maintained in patients with hypothyroidism by administering LT4 suppositories at a dose 1.8 times higher than that of the tablet. Thus, the administration of LT4 suppository can be an alternative for treatment with oral medication in clinical practice.