STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages.