Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-gamma signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-gamma-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-gamma-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-gamma stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-gamma signaling by targeting STAT1 for ubiquitination and proteasomal degradation.