We previously identified a guanosine triphosphatase (GTPase)-activating protein (GAP) male germ cell Rac GAP (MgcRacGAP) that enhanced interleukin-6 (IL-6)-induced macrophage differentiation of murine M1 leukemia cells. Later, MgcRacGAP was found to play crucial roles in cell division. However, how MgcRacGAP enhanced IL-6-induced differentiation remained elusive. Here we show that MgcRacGAP enhances IL-6-induced differentiation through enhancement of signal transducer and activator of transcription-3 (STAT3) activation. MgcRacGAP, Rac, and STAT3 formed a complex in IL-6-stimulated M1 cells, where MgcRacGAP interacted with Rac1 and STAT3 through its cysteine-rich domain and GAP domain. In reporter assays, the wild-type MgcRacGAP enhanced transcriptional activation of STAT3 while a GAP-domain deletion mutant (DeltaGAP) did not significantly enhance it, suggesting that the GAP domain was required for enhancement of STAT3-dependent transcription. Intriguingly, M1 cells expressing DeltaGAP had no effect on the differentiation signal of IL-6, while forced expression of MgcRacGAP rendered M1 cells hyperresponsive to the IL-6-induced differentiation. Moreover, knockdown of MgcRacGAP by RNA interference profoundly suppressed STAT3 activation, implicating MgcRacGAP in the STAT3-dependent transcription. All together, our data not only reveal an important role for MgcRacGAP in STAT3 activation, but also demonstrate that MgcRacGAP regulates IL-6-induced cellular differentiation in which STAT3 plays a pivotal role.