Ionic hydrogels are biocompatible candidates for skin tissue engineering. Two hydrogels synthesized by crosslinking gelatin with polylysine (positively charged HG1) or polyglutamic acid (negatively charged HG2) were tested using spontaneously immortalized human keratinocytes (HaCaT). HaCaT cells displayed higher adhesion and proliferation onto HG2, forming a continuous and stratified epithelium after 7 days. Moreover HaCaT cells grown onto HG2 showed a decreased Epilysin and Filaggrin expression, while transglutaminase-1 expression was increased. Those data indicate that human keratinocyte can form a stratified epithelium onto HG2 that could therefore be an useful tool for skin tissue engineering.