Background: Cichorium intybus has a wide range of therapeutic applications in Indian traditional systems of medicine, especially in metabolic disorders. Objective: To evaluate the toxicity profile and to investigate the antidiabetic, antihyperlipidemic, and antioxidative efficacy of C. intybus seeds in Wistar rats. Materials and Methods: The aqueous extract of seeds was prepared by decoction, and its quality control analysis was carried out by thin-layer chromatography and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) fingerprinting. Wistar rats were fed with high-fat diet for 5 weeks followed by a single dose of streptozotocin intraperitoneally to induce diabetes. The protective group of rats was given aqueous extract during and after the induction of type 2 diabetes mellitus. Further, repeated dose 28-day (subacute) and repeated dose 90-day (chronic) toxicity studies were conducted as per the OECD guidelines. Results: A total of 18 metabolites have been tentatively identified by UPLC-MS profiling in aqueous extract of C. intybus seeds. No significant changes in mortality and biochemical parameters have been observed during toxicity studies. Moreover, administration of the extract to a protective group of diabetic rats attenuated serum glucose and triglyceride levels by 52.7% and 65.3%, respectively, supported by similar results for parameters related to insulin resistance and oxidative stress. The beneficial effect of extract has also been confirmed through in silico screening. Conclusion: C. intybus can be used as a natural dietary supplement for the prevention and management of diabetes and can be explored to develop a potent phytopharmaceutical for diabetes.