The Warburg effect is detected in numerous types of solid tumors and is characterized by the enhancement of glucose consumption and an increase in the rate of glycolysis in cancer cells. Lactate dehydrogenase A (LDHA) plays a critical role in the Warburg effect and to date, a number of LDHA inhibitors have been identified that act as competitive inhibitors of both the substrate pyruvate and the coenzyme NADH. In our previous study, a set of novel peptides were designed using in silico structure-based method with the aim of inhibiting the activity of LDHA by disrupting the interaction between its enzyme subunits. In the present study employed in vitro and in vivo techniques, to explore the influence of these peptides on the induction of cytotoxicity effect on cancer cell lines. Our findings revealed that the designed peptide effectively inhibits LDHA, leading to the induction of apoptosis and G2/M cell cycle arrest. Finally, we also analyzed the impact of the designed peptide on Balb/c mice, confirming the results of in silico and in vitro studies. Ultimately, the designed peptide had the ability to induce apoptosis in cancer cells by LDHA inhibition.