Aim Krüppel-like factor (KLF) 2 and 4 are two core transcription factors closely related to vascular homeostasis, with multiple protective effects such as anti-inflammatory, anti-calcification and anti-thrombotic. The aim of this study is to elucidate and validate the vascular homeostasis related gene profile co-regulated by KLF2 and KLF4 in endothelial cells. Methods Human umbilical vein endothelial cells (HUVEC) were treated with adenovirus (Ad-KLF2 or Ad-KLF4) and control virus (Ad-NC) for 24 h, RNA was extracted from the cells and analyzed by transcriptomic sequencing. The sequencing results of overexpressed KLF2 and KLF4 were superimposed with the sequencing results of reported KLF2/KLF4 double-gene knockout mice. The selected differential expression genes were verified by real-time fluorescence quantitative PCR in HUVEC treated with Ad-KLF2 or Ad-KLF4, and in HUVEC treated with atorvastatin or resveratrol. Results Transcriptomic superposition revealed 256 differential expression genes were up-regulated by KLF2 and KLF4, and KEGG analysis showed that differential expression genes were enriched in hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, ECM-receptor interaction and focal adhesion; there were 145 differential expression genes down-regulated by KLF2 and KLF4, and KEGG analysis showed that differential expression genes were enriched in microRNA of cancer, mineral absorption, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate, p53 signaling pathway and biosynthesis of amino acids. Finally, six novel genes regulated by KLF2 and KLF4 were obtained. Conclusion FGFR3, SEMA4B, SEMA6A, PTX3, FABP4 and FABP5 may be novel genes that regulate vascular homeostasis in endothelial cells by the transcription factors KLF2 and KLF4.