Loss of gastric acid secretion is pathologically known as achlorhydria. Acid-secreting parietal cells are characterized by abundant expression of ezrin (Vil2), one of ezrin/radixin/moesin proteins, which generally cross-link actin filaments with plasma membrane proteins. Here, we show the direct in vivo involvement of ezrin in gastric acid secretion. Ezrin knockout (Vil2(-/-)) mice did not survive >1.5 wk after birth, making difficult to examine gastric acid secretion. We then generated ezrin knockdown (Vil2(kd/kd)) mice by introducing a neomycin resistance cassette between exons 2 and 3. Vil2(kd/kd) mice born at the expected Mendelian ratio exhibited growth retardation and a high mortality. Approximately 7% of Vil2(kd/kd) mice survived to adulthood. Ezrin protein levels in Vil2(kd/kd) stomachs decreased to <5% of the wild-type levels without compensatory up-regulation of radixin or moesin. Adult Vil2(kd/kd) mice suffered from severe achlorhydria. Immunofluorescence and electron microscopy revealed that this achlorhydria was caused by defects in the formation/expansion of canalicular apical membranes in gastric parietal cells.