Fenugreek [Trigonella foenum graecum (Linn.); family Papilionaceae] is commonly known as Methi in Hindi and Bengali. It is commonly used as a dietary ingredient (spice) in India and other parts of the world. It has long been used for several medicinal purposes in folk medicine. In the present study, trigonelline was isolated from the fenugreek seeds at 99% purity. Oxidative stress was generated, in vitro, by copper-ascorbate in mitochondria isolated from goat aortic tissue and the changes brought about in the levels of biomarkers of oxidative stress, activities of antioxidant and the levels of reactive nitrogen species, activities of Kreb’s cycle enzymes and respiratory chain enzymes, cardiolipin content, di-tyrosine fluorescence, mitochondrial swelling and mitochondrial morphology were studied. When mitochondria were co-incubated with copper-ascorbate and trigonelline [99%] then protections were found against copper-ascorbate induced oxidative stress mediated changes in mitochondria in a dose-dependent manner and antioxidant mechanisms appear to be associated with such protections. The results of the current study suggest that trigonelline [99%] may be considered as a future therapeutic antioxidant for the treatment of diseases associated with mitochondrial oxidative stress. © 2014, Global Research Online. All rights reserved.