Heme biosynthesis pathway is conserved in yeast and humans and hem12 yeast mutants mimic porphyria cutanea tarda (PCT), a hereditary human disease caused by mutations in the UROD gene. Even though mutations in other genes also affect UROD activity and predispose to sporadic PCT, the regulation of UROD is unknown. Here, we used yeast as a model to study regulation of Hem12 by ubiquitination and involvement of Rsp5 ubiquitin ligase in this process. We found that Hem12 is monoubiquitinated in vivo by Rsp5. Hem12 contains three conserved lysine residues located on the protein surface that can potentially be ubiquitinated and lysine K8 is close to the 36-LPEY-39 (PY) motif which binds WW domains of the Rsp5 ligase. The hem12-K8A mutation results in a defect in cell growth on a glycerol medium at 38°C but it does not affect the level of Hem12. The hem12-L36A,P37A mutations which destroy the PY motif result in a more profound growth defect on both, glycerol and glucose-containing media. However, after several passages on the glucose medium, the hem12-L36A,P37A cells adapt to the growth medium owing to higher expression of hem12-L36A,P37A gene and higher stability of the mutant Hem12-L36A,P37A protein. The Hem12 protein is downregulated upon heat stress in a Rsp5-independent way. Thus, Rsp5-dependent Hem12 monoubiquitination is important for its functioning, but not required for its degradation. Since Rsp5 has homologs among the Nedd4 family of ubiquitin ligases in humans, a similar regulation by ubiquitination might be also important for functioning of the human UROD.