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Gamma linolenic acid is a polyunsaturated fatty acid having selective anti-tumour properties with negligible systemic toxicity. In the present study, the anti-cancer potential of gamma linolenic acid and its effects on mitochondrial as well as hypoxia-associated marker was evaluated. The effect of gamma linolenic acid was scrutinised against ER + MCF-7 cells by using fluorescence microscopy, JC-1 staining, dot plot assay and cell cycle analysis. The in vitro results were also confirmed using carcinogen (n-methyl-n-nitrosourea) induced in vivo model. The early and late apoptotic signals in the conjugation with mitochondrial depolarisation were found once scrutinised through mitochondrial membrane potential and life death staining after gamma linolenic acid treatment. Gamma linolenic acid arrested the cell cycle in G0/G1 phase with the majority of cell populations in the early apoptotic stage. The translocation of phosphatidylserine was studied through annexin-V FITC dot plot assay. The markers of cellular proliferation (decreased alveolar bud count, histopathological architecture restoration and loss of tumour micro-vessels) were diminished after gamma linolenic acid treatment. Gamma linolenic acid ameliorates the biological effects of n-methyl-n-nitrosourea persuading the mitochondrial mediated death pathway and impeding the hypoxic microenvironment to make a halt in palmitic acid synthesis. SIGNIFICANCE: The present study elaborates the effect of gamma linolenic acid on mammary gland cancer by following mitochondrial-mediated death apoptosis pathway. Gamma linolenic acid also inhibits cell-wall synthesis by the curtailment of HIF-1α and FASN level in mammary gland cancer.