Purpose Metabolic MRI is a noninvasive technique that can give new insights into understanding cancer metabolism and finding biomarkers to evaluate or monitor treatment plans. Using this technique, a previous study has shown an increase in pH during neoadjuvant chemotherapy (NAC) treatment, while recent observation in a different study showed a reduced amide proton transfer (APT) signal during NAC treatment (negative relation). These findings are counterintuitive, given the known intrinsic positive relation of APT signal to pH. Methods In this study we combined APT MRI and 31P‐MRSI measurements to unravel the relation between the APT signal and pH in breast cancer. Twenty‐two breast cancer patients were scanned with a 7 T MRI before and after the first cycle of NAC treatment. pH was determined by the chemical shift of inorganic phosphate (Pi). Results While APT signals have a positive relation to pH and amide content, we observed a direct negative linear correlation between APT signals and pH in breast tumors in vivo. Conclusions As differentiation of cancer stages was confirmed by observation of a linear correlation between cell proliferation marker PE/Pi (phosphoethanolamine over inorganic phosphate) and pH in the tumor, our data demonstrates that the concentration of mobile proteins likely supersedes the contribution of the exchange rate to the APT signal. 31P‐MRS and CEST‐MRI were acquired in 22 breast cancer patients with a 7 T MRI. While APT signals have a positive relation to pH and amide content, we observed a direct negative linear correlation between APT signals and pH in breast tumors in vivo. Our data demonstrates that the concentration of mobile proteins likely supersedes the contribution of the exchange rate to the APT signal.