The present study identified a novel salinomycin (Sal) sensitization mechanism in cancer. We tested whether Sal reduced proliferation in a high-density population by counting attached cell numbers after Sal treatment. Sal reduced proliferation in high-density cell populations. Longer exposure to Sal further reduced proliferation. Sal concentrations of 0.1 and 5 mu M had similar sensitization effects, suggesting that Sal toxicity was minimal with longer exposure to a high-density cell population. The results suggest that Sal can be applied at a relatively low concentration for a longer time to overcome drug-resistant solid tumors. The 0.5 mu M Sal treatment resulted in fewer attached cells than that of the 5 mu M Sal treatment with a longer exposure. The lower Sal concentration mainly increased the number of easily detachable cells on the surface. In particular, 0.5 mu M Sal increased cellular detachment of newly produced daughter cells. The easily-detachable cells were undergoing apoptosis. It seems that the 0.5 mu M Sal treatment also increased cellular toxicity. These novel findings may contribute to the development of Sal-based therapy for patients with drug-resistant cancer or a high-density solid tumor.