Glucosylceramide (GlcCer) is a signaling lipid involved in the regulation of several cellular processes. It is present in different organelles, including the plasma membrane, Golgi apparatus, endoplasmic reticulum, and lysosomes. Accordingly, GlcCer is exposed to different pH environments in each organelle, which may lead to alterations in its properties and lateral organization and subsequent biological outcome. In this study, we addressed the effect of pH on the biophysical behavior of this lipid and other structurally related sphingolipids (SLs). Membranes composed of POPC (1-palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine) and C16-GlcCer, sphingomyelin, and different acyl chain ceramides were characterized by fluorescence spectroscopy, confocal microscopy, and surface pressure area measurements under neutral and acidic conditions. The results show that changing the pH from 7.4 to 5.5 has a larger impact on C16-GlcCer-containing membranes compared to other SLs. In addition, acidification mainly affects the organization and packing properties of the GlcCer-enriched gel phase, suggesting that the interactions established by the glucose moiety, in the GlcCer molecule, are those most affected by the increase in the acidity. These results further highlight the role of GlcCer as a modulator of membrane biophysical properties and will possibly contribute to the understanding of its biological function in different organelles.