STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.