Objective  To investigate the regulatory effects of intestinal-derived uremic toxin trimethylamine oxide (TMAO) on the JNK/Bcl-2 signaling pathway and the mechanisms of vascular endothelial cell injury in uremic rats.  Methods  Five-week-old SD rats were selected and established as uremia models. The rats were divided into model group, TMAO group (3% TMAO), SP600125 group (15 mg/kg), TMAO+SP group, and sham operation control group (n=12). Body weight, 24-hour urinary protein, serum creatinine (Scr), and blood urea nitrogen (BUN) levels were measured. Histopathological changes were observed using PAS and HE staining. The expressions of endothelial nitric oxide synthase (eNOS) and vascular cell adhesion molecule-1 (VCAM-1) were detected by RT-qPCR and immunohistochemistry. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were measured by ELISA. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were measured. Cell apoptosis was assessed by TUNEL method. The expressions of Bax, Caspase-3, p-JNK, and p-Bcl-2 were determined by western blot.  Results  Compared with the sham operation control group, the model group exhibited elevated Scr, blood BUN, and urinary protein (F=8.640, 13.025 and 10.998 respectively; P＜0.001), decreased eNOS expression and increased VCAM-1 expression (F=21.951, 20.080; P＜0.001), elevated TNF-α, IL-1β, and IL-6 (F=10.678, 32.043 and 21.181 respectively; P＜0.001), reduced SOD and GSH-Px activity with increased MDA level (F=14.172, 11.445 and 13.984 respectively; P＜0.001), and increased TUNEL positivity rate along with elevated Bax, caspase-3, p-JNK, and p-Bcl-2 (F=36.357, 12.926, 10.092, 20.842 and 19.200 respectively; P＜0.001). TMAO further exacerbated these pathological changes. SP600125 reversed the pathological changes in model group as described above, including reduced Scr, BUN, and urinary protein (F=8.640, 13.025 and 10.998 respectively; P＜0.001), upregulated eNOS expression and downregulated VCAM-1 expression (F=21.951, 20.080; P＜0.001), decreased TNF-α, IL-1β, and IL-6 (F=10.678, 32.043 and 21.181 respectively; P＜0.001), increased the activities of SOD and GSH-Px and reduced MDA level (F=14.172, 11.445 and 13.984 respectively; P＜0.001), decreased TUNEL positivity rate and the expressions of Bax, Caspase-3, p-JNK, and p-Bcl-2 (F=36.357, 12.926, 10.092, 20.842 and 19.200 respectively; P＜0.001).  Conclusion TMAO aggravates vascular endothelial cell injury in uremic rats by activating the JNK/Bcl-2 signaling pathway, thereby promoting inflammatory responses, oxidative stress, and apoptosis.