Type I interferon (IFN-I) is a pluripotent cytokine that modulates the innate and adaptive immunity. IFN-I activates its downstream signal mediators, including STAT1, STAT2 and STAT3. While STAT1 and STAT2 are positive regulators of IFN-I response, STAT3 is, on the contrary, a negative regulator. However, the molecular mechanisms of the suppressive effect of STAT3 remain unclear. We have previously shown that STAT3 inhibited IFN-I-mediated ISG expression and antiviral activity in a manner dependent on its N-terminal domain (NTD) but independent of its DNA binding and transactivation ability. Using yeast two-hybrid system, we have identified phospholipid scramblase 2 (PLSCR2) as a STAT3-interacting protein and a suppressor of IFN-I response. PLSCR2 mainly located in the nucleus and was an IFN-I inducible gene. Overexpression of full length or C-terminal domain of PLSCR2 attenuated ISRE-driven reporter activities, which was further aggravated by STAT3 coexpression. Knockdown or knockout of PLSCR2 enhanced IFN-induced ISG induction and antiviral response without affecting the activation or nuclear translocation of STAT1 and/or STAT2. Instead, the assembly and recruitment of ISGF3 to the promoters of ISGs were enhanced, suggesting a negatively regulatory role of PLSCR2 on IFN-I. Interestingly, both gain- and loss-of-function activity of PLSCR2 was STAT3-dependent. PLSCR2 interacted with STAT3 NTD through its palmitoylation motif. Functional mutations of the motif ablated the interaction with STAT3, leading to blockage of the suppressive activities of PLSCR2. In sum, these results define PLSCR2 as a novel STAT3-dependent, negative regulator of IFN-I response.