Thymoquinone (TQ), a naturally occurring anticancer compound extracted from Nigella sativa oil, has been extensively reported to possess potent anti-cancer properties. Experimental studies showed the anti-proliferative, pro-apoptotic, and anti-metastatic effects of TQ on different cancer cells. One of the possible mechanisms underlying these effects includes alteration in key metabolic pathways that are critical for cancer cell survival. However, an extensive landscape of the metabolites altered by TQ in cancer cells remains elusive. Here, we performed an untargeted metabolomics study using leukemic cancer cell lines during treatment with TQ and found alteration in approximately 335 metabolites. Pathway analysis showed alteration in key metabolic pathways like TCA cycle, amino acid metabolism, sphingolipid metabolism and nucleotide metabolism, which are critical for leukemic cell survival and death. We found a dramatic increase in metabolites like thymine glycol in TQ-treated cancer cells, a metabolite known to induce DNA damage and apoptosis. Similarly, we observed a sharp decline in cellular guanine levels, important for leukemic cancer cell survival. Overall, we provided an extensive metabolic landscape of leukemic cancer cells and identified the key metabolites and pathways altered, which could be critical and responsible for the anti-proliferative function of TQ.