Transcription factors (TFs) are modulators of gene expression that are critically important in the establishment and progression of human cancers. In the current study, the activity profiles of TFs in a normal nasopharyngeal epithelial cell line and in nasopharyngeal carcinoma (NPC) cell lines were studied using oligonucleotide array-based TF assays. Compared to the normal epithelial cell line NP69, nine TFs in the non-metastatic NPC cell line (6-10B) and eight TFs in a metastatic NPC cell line (5-8F) were upregulated. Among upregulated TFs, Sp1, AP2, and ATF/CREB families exhibited relatively high activities in NPC cell lines. Transcription levels of Sp1, ATF-1, ATF-2, AP2alpha, AP2gamma, and CREB1 were higher in 5-8F cells than in 6-10B cells. In addition, higher expression of the Sp1 target genes MMP-9 and VEGF was observed in 5-8F cells. Sp1 silencing reduced VEGF and MMP-9 expression. Inhibition of Sp1 expression and activity in 5-8F cells by mithramycin resulted in downregulated expression and secretion of MMP-9 and VEGF, concomitant with inhibition of cell migration and invasion. These results suggest that dynamic changes in TF activities occur in NPC cells and that these changes may play important roles in regulating the expression of genes associated with the development and progression of NPC.