Reactive oxygen species (ROS), playing a two-fold role in tumorigenesis, are responsible for tumor formation and progression through the induction of genome instability and pro-oncogenic signaling. The same ROS is toxic to cancer cells at higher levels, oxidizing free nucleotide precursors (dNTPs) as well as damaging DNA leading to cell senescence. Research has highlighted the tumor cell-specific expression of a redox-protective phosphatase, MutT homolog 1 (MTH1), that performs the enzymatic conversion of oxidized nucleotides (like 8-oxo-dGTP) to their corresponding monophosphates, up-regulated in numerous cancers, circumventing their misincorporation into the genomic DNA and preventing damage and cell death. To identify novel natural small molecular inhibitors of MTH1 to be used as cancer therapeutic agents, molecular screening for MTH1 active site binders was performed from natural small molecular libraries. Emodin was identified as a lead compound for MTH1 active site functional inhibition and its action on MTH1 inhibition was validated on non-small cell lung cancer cellular models (NSCLC). Our study provides strong evidence that emodin mediated MTH1 inhibition impaired NSCLC cell growth, inducing senescence. Emodin treatment enhanced the cellular ROS burdens, on one hand, damaged dNTP pools and inhibited MTH1 function on the other. Our work on emodin indicates that ROS is the key driver of cancer cell-specific increased DNA damage and apoptosis upon MTH1 inhibition. Consequently, we observed a time-dependent increase in NSCL cancer cell susceptibility to oxidative stress with emodin treatment. Based on our data, the anti-cancer effects of emodin as an MTH1 inhibitor have clinical potential as a single agent capable of functioning as a ROS inducer and simultaneous blocker of dNTP pool sanitation in the treatment of NSCL cancers. Collectively, our results have identified for the first time that the potential molecular mechanism of emodin function, increasing DNA damage and apoptosis in cancer cells, is via MTH1 inhibition.