Precise regulation of extracellular pH is critical for proper function of many cellular activities. Minor fluctuations in pH can have profound effects on ion transport, receptor function, and cellular secretions. This work examines the impact of acidic pH, universal at sites of inflammation, on the activation status of mast cells. The mast cell line HMC-1 was used to explore activation by acidic pH. Activation of HMC-1 cells was characterized in cell adhesion, secretion of cytokines, and changes in cell morphology. HMC-1 adhesion to two substrates, collagen and laminin was measured at 0, 1 hr, 2 hrs, 4 hrs, and 6 hrs. To determine the effect of acidic pH on adhesion, a comparison was made between adhesion in normal physiological pH (pH 7.4) and acidic pH (pH 6.5). The secretion of cytokines by HMC-1 was examined using a 42 cytokine array, results were quantified with ELISAs. Changes in the morphology of HMC-1 were examined with scanning electron microscopy. The involvement of a pH receptor from the TRPV family was tested by application of specific blockers. A shift to acidic pH (pH 6.5) initiated an increase in adhesion that was time, and dose dependent, and with a preference for collagen. HMC-1 secretion of the cytokine MCP-1 decreased in response to exposure to acidic pH. The receptor TRPV1 is implicated as playing a role in the response of HMC-1 to acidic pH with the finding that adhesion decreased and MCP-1 secretion increased in the presence of the TRPV1 antagonist ruthenium red. The shift to acidic pH also resulted in significant morphological changes in the morphology of HMC-1. In summary, acidic pH induces mast cells to adhere to extracellular matrix proteins, secrete cytokines as well as restructure the cytoskeleton. This study helps to define the functional responses of mast cells to acidic pH.