KLF1/EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the HBB-like globin genes. Prompted by the observation that four KLF sites are distributed in the human alpha-globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four alpha-globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4-specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4-DNA binding, were transactivated by over-expression of KLF4. In K562 cells, induced KLF4 expression with a Tet-off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh-RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.