Abstract Background Standard biological therapy for moderate-severe cases of UC consists in the use of Infliximab (IFX), an anti-tumour necrosis factor α (anti-TNF-α) targeting agent. However, approximately 40% of the patients do not respond to IFX. While transcriptional alterations have been widely associated with non-response to IFX, the precise factors that regulates this altered gene expression remains poorly understood. The aim of this study was perform a robust in silico analysis to identify master transcriptional regulators (MTRs) which regulate gene expression changes in IFX non-responsive UC patients, followed by perturbation in vitro to establish their functional role in UC pathogenesis. Methods Differentially expressed genes (DEGs) identified from four independent public datasets were applied to the GeneXplain platform to identify transcription factors (TFs) enriched for the DEG’s. This was followed by an upstream analysis to identify MTR’s which regulate expression of these genes through the TFs. We next explored the role of these MTRs in driving UC-associated phenotype, using siRNA-mediated knock-down in a novel IBD-like in vitro system comprising of normal rectal epithelial cells treated with TNFa. Results Over 200 TFs were identified in both upregulated and downregulated genes in IFX non-responders across all data sets. RELA, NANOG, FOSJUN were the top 3 TF’ enriched for upregulated genes in three out of the four datasets. Using the TF data, the upstream analysis identified CXCL8, SELE, PTGS2, FCGR3, TFPI2 as the top five MTRs. Next, we showed that of the 5 MTRs, only TFPI2 (tissue factor pathway inhibitor 2) were significantly upregulated in an IBD-like in vitro model (normal rectal epithelial cell line: CRL1831 +TNFa). siRNA-mediated knock-down of TFPI2 in this model resulted in a significant decrease in genes including TNFAIP6, TNFAIP3, FCGR3B, BIRC3 and CXCL5, all of which were predicted in silico to be upregulated by TFPI2 during the upstream analysis. Intriguingly, we showed that downregulation of TFPI2 resulted in significant amelioration of pro-inflammatory cytokines including IL1β, IFNγ, IL10, IL13, IL6 and IL8. Conclusion Our study for the first time identifies key MTRs such TFPI2 which acts as a master regulator of genes whose upregulation drives poor response to IFX. Therefore, these results warrant further in vitro/ex vivo investigation to rationalise their role of TFPI2 as a potential therapeutic target, whose perturbation in UC patient would likely result in improved response to IFX.