Maintenance of a neutral intracellular pH (pHi) is favorable for the survival of tumors, and maintenance of highly acidic extracellular pH (pHe) facilitates tumor invasiveness. The aim of the present study was to investigate the antitumor effects of lactate calcium salt (CaLa), 5-indanesulfonamide (IS) and α-cyano-4-hydroxycinnamic acid (CA) via pH regulation in colon cancer cells. HCT116 cells were treated with CaLa, IS, CA and combinations of the three. Subsequently, the concentration of intracellular lactate was determined. pHi and pHe were measured using cell lysates and culture media. Colony formation assay, cell viability assay and western blot analysis were additionally performed to analyze the consequences of the pH changes. CaLa, IS, CA and combination treatments induced an increase in the concentration of intracellular lactate. Lactate influx into the tumor microenvironment produced an acidic pHi in colon cancer cells. Consequently, colony formation and cell viability were significantly decreased, as well as poly(adenosine diphosphate-ribose) polymerase degradation. The tumor microenvironment may be exploited therapeutically by disrupting the mechanism that regulates pHi, leading to cell apoptosis. The present study indicated that treatment with CaLa, IS and CA induced intracellular acidification via lactate influx, causing apoptosis of colon cancer cells. Additionally, the findings suggested that the combination of CaLa with IS and CA may enhance antitumor activity, and may provide a potential therapeutic approach for the treatment of colon cancer.