STAT3 dimers, once activated through phosphorylation at Y705, accumulate in the nucleus for transcriptional activation of its target genes. However, how activated STAT3 dimers are retained in the nucleus and inactivated for nuclear export remains elusive. With using stat3KO-HepG2 (SKO) and stat3- and tc45-double KO-HepG2 (DKO) cells, reconstituted with various Flag-STAT3s, we find that STAT3 accumulates in the nucleus after loss of pY705 in SKO cells but shows early nuclear export despite pY705 in pS727-dependent manner in DKO cells. We show that a kinase activity sensitive to Staurosporine (ST) acts to keep STAT3 in the nucleus. By using leptomycin B, a CRM1 inhibitor, for restricted time together with a system by which we can examine the post-inactivation nuclear export of STAT3 mutants in a short period time, we find that inactivated STAT3 dimer sequentially uses two nuclear export systems, CRM1-dependent and CRM1-independent, depending on the two types of conformation which are regulated by an ST-sensitive kinase activity. Thus, our study shows a novel model for the fate decision of activated nuclear STAT3 from nuclear accumulation to nuclear export, which is regulated by pS727-dependent event(s) and an ST-sensitive kinase activity in a conformation-dependent manner toward the final inactivation form for CRM1-independent nuclear export.