Background: Very little is known about Esophageal squamous cell carcinoma (ESCC),and there is no study systematically investigated its transcriptional regulatory network recently.Materials and Methods: Paired ESCC and non-cancerous (NC) tissues were assayed by Affymetrix microarray chip.Passing Attributes between Networks for Data Assimilation (PANDA) was used to construct networks between transcription factors (TFs) and their targets.AnaPANDA program was applied for comparing regulatory networks.Hypergeometric distribution model based target profile similarity analysis was used to find co-activation effects using both TF-targets networks data and different expression data.Results: There were 1116 genes up regulated and 1301 genes down regulated in ESCC compared with NC.In TF-targets networks,16970 ESCC-specific edges and 9307 NC-specific edges were identified.Edge enrichment analysis by AnaPANDA indicated 17 transcription factors (TP53,NFE2L2,PAX6,TLX1,TBP,STAT1,STAT3,ESRI,NHLHI,ELK4,ELK1,FOXL1,ZNF143,HOXA5) repressed in ESCC and 5 (SPIB,BRCA1,MZF1,MAFG,NFE2L1) activated in ESCC.For SPIB,MZF1,MAFG and NFE2L1,a strong and significant co-activation effect among them was identified in ESCC.Conclusions: Construction of transcriptional regulatory networks found SPIB,MZF 1,MAFG and NFE2L1 co-activated in ESCC,which gives a distinctive insight into carcinogenesis mechanism of ESCC.