TNFα has been identified as playing an important role in pathologic complications associated with diabetic retinopathy and retinal inflammation, such as retinal leukostasis. However, the transcriptional effects of TNFα on retinal microvascular endothelial cells and the different signaling pathways involved are not yet fully understood. In the present study, RNA-seq was used to profile the transcriptome of human retinal microvascular endothelial cells (HRMEC) treated for 4 hours with TNFα in the presence or absence of the NFAT-specific inhibitor INCA-6, in order to gain insight into the specific effects of TNFα on RMEC and identify any involvement of NFAT signaling. Differential expression analysis revealed that TNFα treatment significantly upregulated the expression of 579 genes when compared to vehicle-treated controls, and subsequent pathway analysis revealed a TNFα-induced enrichment of transcripts associated with cytokine-cytokine receptor interactions, cell adhesion molecules, and leukocyte transendothelial migration. Differential expression analysis comparing TNFα-treated cells to those co-treated with INCA-6 revealed 10 genes whose expression was significantly reduced by the NFAT inhibitor, including those encoding the proteins VCAM1 and CX3CL1 and cytokines CXCL10 and CXCL11. This study identifies the transcriptional effects of TNFα on HRMEC, highlighting its involvement in multiple pathways that contribute to retinal leukostasis, and identifying a previously unknown role for NFAT-signaling downstream of TNFα.