Stratum corneum (SC) is the outermost layer of the epidermis, which consists of nonviable anuclear keratinocytes (corneocytes) and function as a protective skin barrier. The pH distribution of SC has long been considered to impact its homeostasis. However, pH distribution and its biological significance in SC homeostasis remain unclear due to challenges in measuring and visualizing pH at a single-corneocyte resolution and elucidating the biological significance of pH profiles in SC. To tackle these challenges, we generated mice expressing Venus–mCherry, the fusion protein of pH-sensitive probe Venus and pH-insensitive probe mCherry from the uppermost stratum granulosum cells (SASP-Venus-mCherry mice), which enables the observation of relative pH changes and calculation of pH values via simultaneous ratiometric imaging. Confocal microscopic analysis of SASP-Venus-mCherry mice revealed that the SC has three stepwise pH zones, lower-moderately acidic (pH 6.0), middle-acidic (pH 5.4), and upper-nearly neutral (pH 6.7) zones, rather than gradual change over SC layers, in various body parts. Topical application of phosphate buffers with pH 4.1, 5.3, 6.4 and 7.2 did not affect pH of lower and middle SC-pH zones, while it changed pH of upper SC-pH zone according to the applied buffer. Additionally, the lower and middle SC-pH zones were dependent on claudin-1, a tight junction protein, which diminished in claudin-1-deficient mice; thus, the entire SC exhibits neutral pH. Finally, mathematical modeling demonstrated that the stepwise SC-pH profile pH6.0–pH5.4–pH6.7, but not the gradient SC-pH profile pH7–pH5 reproduced the increase of effective catalytic activity of kallikrein-related peptidases toward the SC surface. Collectively, these findings indicate that the three stepwise SC-pH zones are tightly regulated and are necessary to mediate adequate desquamation, which leads to functional SC differentiation.