Gastric proton pump (H⁺,K⁺-ATPase) consists of two subunits, a catalytic α-subunit and a glycosylated β-subunit (βHK). So far, properties of the individual carbohydrate residues of βHK have been unclear. Here, we succeeded in visualizing the sialylation and desialylation dynamics of βHK using a fluorescence bioimaging nanoprobe that specifically detects sialic acids. The fluorescence of the probe was observed at the cell surface of H⁺,K⁺-ATPase-expressing living LLC-PK1 cells but not in non-expressing cells. The fluorescence and H⁺,K⁺-ATPase activity in the cells were significantly decreased by sialidase and acidic solution. In gastric mucosa of rats and hogs, the fluorescence of the probe was observed in the samples treated by famotidine, an H₂ blocker, but not by histamine, an acid secretagogue. H⁺,K⁺-ATPase activity in the famotidine-treated samples was significantly higher than the histamine-treated samples. These famotidine-induced effects were weakened by sialidase. Our studies using the nanoprobe uncover a novel negative-feedback mechanism of H⁺,K⁺-ATPase in which sialic acids of βHK positively regulates H⁺,K⁺-ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of βHK.