Hydroxyurea (HU) is effectively used in the management of β-hemoglobinopathies by augmenting the production of fetal hemoglobin (HbF). However, the molecular mechanisms underlying HU-mediated HbF regulation remain unclear. We previously reported that overexpression of the HU-induced SAR1 gene closely mimics the known effects of HU on K562 and CD34(+) cells, including γ-globin induction and cell-cycle regulation. Here, we show that HU stimulated nuclear factor-κB interaction with its cognate-binding site on the SAR1 promoter to regulate transcriptional expression of SAR1 in K562 and CD34(+) cells. Silencing SAR1 expression not only significantly lowered both basal and HU-elicited HbF production in K562 and CD34(+) cells, but also significantly reduced HU-mediated S-phase cell-cycle arrest and apoptosis in K562 cells. Inhibition of c-Jun N-terminal kinase (JNK)/Jun phosphorylation and silencing of Giα expression in SAR1-transfected K562 and CD34(+) cells reduced both γ-globin expression and HbF level, indicating that activation of Giα/JNK/Jun proteins is required for SAR1-mediated HbF induction. Furthermore, reciprocal coimmunoprecipitation assays revealed an association between forcibly expressed SAR1 and Giα2 or Giα3 proteins in both K562 and nonerythroid cells. These results indicate that HU induces SAR1, which in turn activates γ-globin expression, predominantly through the Giα/JNK/Jun pathway. Our findings identify SAR1 as an alternative therapeutic target for β-globin disorders.