Overexpression of human ribonucleotide reductase subunit M2 (hRRM2) has been shown as a potential factor causing Gemcitabine (Gem) resistance. We hypothesized the nuclear factor Y (NF-Y) would transcriptionally regulate hRRM2 and contribute to overexpression of hRRM2 in a Gem resistant clone. A luciferase and gel shift assay, and a Southwestern blot were employed to analyze the promoter activity of hRRM2. The data exhibited the hRRM2 promoter was upregulated almost 5-fold in the Gem resistant KB clone (KBGem) via three sequential CCAAT boxes located in the proximal promoter region. Nuclear extracts from KB and KBGem could interact with the CCAAT motif of the hRRM2 proximal promoter region, and could form DNA-protein complexes with different binding patterns. The complexes could be further recognized with antibodies against NF-Y subunits A and B. Histone deacetylases (HDAC) involvement in NF-Y transcription repression in the KBGem clone was examined. A HDAC activity assay revealed a 3-fold decrease of HDAC activity in the KBGem clone compared to KB cells. Parental cells were treated with trichostatin A (TSA), a HDAC inhibitor. NF-Y transactivation was induced, resulting in an increase of hRRM2 expression. This led to an expanded dCTP pool and an abrogated [3H]Gemcitabine incorporation. In addition, microarray analysis results showed most of the proliferation-related genes were upregulated in KBGem. This finding was consistent with enhanced NF-Y transactivation in KBGem. In summary, upregulation of NF-Y transactivation increased hRRM2 transcription, which played a pivotal role in the Gem resistant KB clone.