When intracellular reactive oxygen species (ROS) increase, cancer cells are more vulnerable to oxidative stress compared to normal cells; thus, the collapse of redox homeostasis can lead to selective death of cancer cells. Indeed, recent studies have shown that inhibition of sulfiredoxin (Srx), which participates in antioxidant mechanisms, induces ROS-mediated cancer cell death. In this paper, we describe how an Srx inhibitor, J14 (4-[[[4-[4-(2-chlor-ophenyl)-1-piperazinyl]-6-phenyl-2-pyrimidinyl]thio]methyl]-benzoic acid), interferes with the antioxidant activity of Srx at the molecular level. We searched for possible binding sites of Srx using a binding site prediction method and uncovered two possible inhibition mechanisms of Srx by J14. Using molecular dynamics simulations and binding free energy calculations, we confirmed that J14 binds to the ATP binding site; therefore, J14 acts as a competitive inhibitor of ATP, settling the question of the two mechanisms. Based on the inhibition mechanism revealed at the atomic level, we designed several derivatives of J14, which led to LMT-328 (4-(((4-(4-(2-Chlorophenyl)piperazin-1-yl)-6-(2,4-dihydroxy-5-isopropylphenyl)pyrimidin-2-yl)thio)methyl)benzoic acid), which is possibly an even more potent inhibitor than J14.