Aloe vera is a popular medicinal plant in the cosmetic, pharmaceutical, and food industries. Acemannan (ACE), a β-(1,4)-acetylated mannan, is one of the bioactive compounds isolated from the A. vera gel. The pharmacological effects of ACE have been reported regarding digestive disease protection, antimicrobia, and prebiotic activity. Here, we used human HaCaT cells as a model to uncover the potential biological functions of ACE in keratinocytes. ACE increased cell growth in a concentration-dependent manner, and a higher incorporation of BrdU was detected in ACE-treated cells than in vehicle-treated cells, indicating ACE promotes cell proliferation. Furthermore, ACE concentration-dependently promoted cell migration in the wound scratch model. ACE regulated cell differentiation by transiently decreasing p63α expression, but increasing the expression of involucrin, loricrin, and transglutaminase 1 (TGase 1). These effects were non-additive to those induced by phorbol myristate acetate (PMA), but additive to epidermal growth factor (EGF), which are complete and incomplete differentiation agents of keratinocytes, respectively. Moreover, ACE activated EGF receptor (EGFR), protein kinase C (PKC), and protein kinase B (AKT/PKB). PKC inhibitor Ro320432 enhanced cell growth and migration, while EGFR inhibitor osimertinib blocked both responses. In summary, ACE is a potential therapeutic agent in wound healing. ACE activates PKC, leading to keratinocyte differentiation and activates EGFR, contributing to keratinocyte proliferation and migration.