Hyaluronic acid (HA) is known to play an important role in motility of tumor cells. However, the molecular mechanisms associated with HA-promoted melanoma cell motility are not fully understood. Treatment of cells with HA was shown to increase the production of reactive oxygen species (ROS) in a CD44-dependent manner. Antioxidants, such as N-acetyl-L-cysteine and seleno-L-methionine, prevented HA from enhancing cell motility. Protein kinase C (PKC)-α and PKCδ were responsible for increased Rac1 activity, production of ROS, and mediated HA-promoted cell motility. HA increased Rac1 activity via CD44, PKCα, and PKCδ. Transfection with dominant negative and constitutive active Rac1 mutants demonstrated that Rac1 was responsible for the increased production of ROS and cell motility by HA. Inhibition of NADPH oxidase by diphenylene iodonium and down-regulation of p47Phox and p67Phox decreased the ROS level, suggesting that NADPH oxidase is the main source of ROS production. Rac1 increased phosphorylation of FAK. FAK functions downstream of and is necessary for HA-promoted cell motility. Secretion and expression of MMP-2 were increased by treatment with HA via the action of PKCα, PKCδ, and Rac1 and the production of ROS and FAK. Ilomastat, an inhibitor of MMP-2, exerted a negative effect on HA-promoted cell motility. HA increased interaction between CD44 and epidermal growth factor receptor (EGFR). AG1478, an inhibitor of EGFR, decreased phosphorylation of PKCα, PKCδ, and Rac1 activity and suppressed induction of p47Phox and p67Phox. These results suggest that CD44-EGFR interaction is necessary for HA-promoted cell motility by regulating PKC signaling. EGFR-Akt interaction promoted by HA was responsible for the increased production of ROS and HA-promoted cell motility. In summary, HA promotes CD44-EGFR interaction, which in turn activates PKC signaling, involving Akt, Rac1, Phox, and the production of ROS, FAK, and MMP-2, to enhance melanoma cell motility.