Background: Though the saphenous vein is one of the most commonly used conduits in coronary artery bypass grafting (CABG), the short-term and long-term patency of vein grafts remains relatively low. The effects of cyclic stretch on the transcriptome profile of the saphenous vein and the role of transcription factors (TFs) have not been elucidated. Methods: Baseline information and the excess saphenous veins from 16 patients undergoing elective coronary artery bypass grafting were obtained. The patients were classified as normal control (NC) group and cyclic stretch (ST) group, according to manipulation procedures at harvest. Total RNA was extracted and RNA-seq (RNA sequencing, Transcriptome sequencing technology) performed to investigate changes in transcriptome profiles of venous samples that underwent cyclic stretch. Differentially expressed genes (DEGs), gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) enrichment were performed to study their potential function and pathways. We then used online databases to identify TFs and construct a protein-protein interaction (PPI) network, and identify hub TFs by Cytoscape software. Finally, hub TFs were validated using quantitative reverse transcription PCR (RT-qPCR), immunofluorescence, and western blot. Results: After cyclic stretch, 107 DEGs were identified, including 82 upregulated and 25 downregulated genes. The enrichment of graphene oxide suggests that DEGs are mainly enriched in cellular responses to mechanical stimuli and transcriptional activity. Then, nine differentially expressed TFs were identified after crossing the Encyclopedia of DNA Elements (ENCODE) database with DEGs, and six hub TFs were identified by Cytoscape software and its plug-in CytoHubba, including Early Growth Response Factor 3 (EGR3), activating transcription factor 3 (ATF3), transcription factor (JUN), Mouse Jun B oncogene (JUNB), Finkel Biskis Jinkins (FBJ) mouse osteosarcoma virus oncogenic gene homolog (FOS), and Finkel Biskis Jinkins (FBJ) mouse osteosarcoma virus oncogenic gene homolog B (FOSB). RT-qPCR demonstrated that ATF3 was significantly upregulated in the saphenous vein after cyclic stretch. Western blot further validated those results. Furthermore, immunofluorescence showed that the upregulated ATF3 was predominantly expressed in the tunica media of the saphenous vein. Conclusions: TFs were upregulated in the saphenous vein after cyclic stretch. This may regulate cellular responses to stimuli by regulating transcriptional activity. ATF3 was significantly upregulated in the tunica media of the saphenous vein, indicating it might be a potential biomarker and regulator of smooth muscle cell (SMC) activation caused by cyclic stretch.