Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit <b>9a</b>), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound <b>18n</b> with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with <b>18n</b> resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of <b>18n</b> allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, <b>18n</b> represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.