(1) Background: The acidosis of the tumor micro-environment may have profound impact on cancer progression and on the efficacy of treatments. In the present study, we evaluated the impact of a treatment with UK-5099, a mitochondrial pyruvate carrier (MPC) inhibitor on tumor extracellular pH (pHe); (2) Methods: glucose consumption, lactate secretion and extracellular acidification rate (ECAR) were measured in vitro after exposure of cervix cancer SiHa cells and breast cancer 4T1 cells to UK-5099 (10 µM). Mice bearing the 4T1 tumor model were treated daily during four days with UK-5099 (3 mg/kg). The pHe was evaluated in vivo using either chemical exchange saturation transfer (CEST)-MRI with iopamidol as pHe reporter probe or P-NMR spectroscopy with 3-aminopropylphosphonate (3-APP). MR protocols were applied before and after 4 days of treatment; (3) Results: glucose consumption, lactate release and ECAR were increased in both cell lines after UK-5099 exposure. CEST-MRI showed a significant decrease in tumor pHe of 0.22 units in UK-5099-treated mice while there was no change over time for mice treated with the vehicle. Parametric images showed a large heterogeneity in response with 16% of voxels shifting to pHe values under 7.0. In contrast, P-NMR spectroscopy was unable to detect any significant variation in pHe; (4) Conclusions: MPC inhibition led to a moderate acidification of the extracellular medium in vivo. CEST-MRI provided high resolution parametric images (0.44 µL/voxel) of pHe highlighting the heterogeneity of response within the tumor when exposed to UK-5099.