Smad ubiquitin regulatory factor 2 (Smurf2) is a HECT domain-containing E3 ubiquitin ligase. Together with its closely related homolog Smurf1, Smurf2 was initially recognized as a negative regulator of transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling, but subsequent studies have expanded its function to regulate many different signaling pathways and play important roles in genomic stability, cell polarity, tissue homeostasis and carcinogenesis. Mice with conventional knockout of Smurf1 or Smurf2 alleles are viable, but conventional Smurf1 and Smurf2 double knockout mice were early embryonic lethal. In order to study the physiological function of Smurfs during late stage of embryonic development or in adult animals, we generated Smurf2 mice carrying a targeted mutation for conditional Smurf2 gene inactivation. We demonstrated that Cre-mediated recombination using Alb-Cre, a Cre line expressed in hepatocyte, results in specific deletion of the gene in liver tissue. We also showed that Cre-mediated recombination in mouse embryonic fibroblasts (MEFs) with Smurf2 genotype resulted in generation of Smurf2 knockout MEFs, and Smurf2 deficiency affects multiple signaling pathways. Therefore, this animal model will be useful to study the distinct roles of Smurf2 in different tissues at different ages.