Human type 2 iodothyronine deiodinase (NOREF>hD2) catalyzes the activation of T₄ to T₃. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec¹³³ to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec¹³³. The K_m (T₄) of Cys¹³³ D2 was 2.1 μm, strikingly higher than that of native D2 (1.4 nm). In contrast, the relative turnover number was 10-fold lower for Cys¹³³D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys¹³³D2 enzyme exhibited saturation kinetics expected from the K_m as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T₄. Blockade of the NTCP, OATP1–3, and LST-1 transporters with 10 mm sodium taurocholate did not alter the deiodination rate of T₄ by Cys¹³³D2 transiently expressed in intact cells, suggesting that intracellular transport of T₄ is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.