STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine‐phosphorylated transcription factor. Here, we identify and characterize a non‐transferable nuclear targeting sequence in the STAT1 DNA‐binding domain. This conserved signal is critical for the interferon‐γ (IFN‐γ)‐induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN‐γ stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss‐of‐function mutations of the dimer‐specific import signal block nuclear entry of tyrosine‐phosphorylated STAT1, which in turn also prevents induction of cytokine‐inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1‐dependent constitutive expression of caspases and the tumor necrosis factor‐α‐mediated induction of apoptosis proceed unaltered. Thus, tyrosine‐phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes.