Proton pump inhibitors reduce gastric acid secretion and are commonly utilized in the management of gastroesophageal reflux disease (GERD) across all ages. Yet a decrease in lower esophageal sphincter tone has been reported in vitro in rats through an unknown mechanism, however their effect on the gastroesophageal muscle tone early in life was never studied. Hypothesizing that proton pump inhibitors also reduce gastroesophageal muscle contraction in newborn and juvenile rats, we evaluated the in vitro effect of pantoprazole on gastric and lower esophageal sphincter muscle tissue. Electrical field stimulation (EFS) and carbachol-induced force were significantly (P<0.01) reduced in the presence of pantoprazole, whereas the drug had no effect on the neuromuscular-dependent relaxation. When administered in vivo, pantoprazole (9 mg/kg) significantly (P<0.01) reduced gastric emptying time at both ages. In order to ascertain the signal transduction pathway responsible for the reduction in muscle contraction, we evaluated the tissue rho-associated kinase 2 (ROCK-2) and CPI-17 activity. Pantoprazole reduced myosin light chain phosphatase MYPT-1, but not CPI-17 phosphorylation of gastric and lower esophageal sphincter tissue strongly suggesting that it is a ROCK-2 inhibitor. To the extent that these findings can be extrapolated to human neonates, the use of pantoprazole may impair gastric and lower sphincter muscle tone and thus paradoxically exacerbate esophageal reflux. Further studies addressing the effect of proton pump inhibitors on gastroesophageal muscle contraction are warranted to justify its therapeutic use in GERD.