The aim of this paper was to investigate the effect of terpene penetration enhancers on membrane fluidity and membrane potential using HaCaT keratinocytes, and study the potential mechanisms of these terpene compounds using as natural transdermal penetration enhancer. Six terpene compounds, namely menthol, limonene, 1,8-cineole, menthone, terpinen-4-ol and pulegone, were chosen in this study on account of their good penetration-enhancement activities. The cytotoxicity of these terpene compounds was measured using an MTT assay. The fluorescence recovery after photobleaching (FRAP) technique was employed to measure the change of membrane fluidity of HaCaT cells. The flow cytometer was used to study the alteration of membrane fluidity of HaCaT cells, and investigate the effect of terpene compounds on intracellular Ca2+. It was found that 6 terpene compounds possessed low cytotoxicity in comparison to the well-established and standard penetration enhancer azone. Those terpene compounds could significantly enhance HaCaT cells membrane fluidity and decrease HaCaT cells membrane potentials. Meanwhile, after treated with various terpene compounds, the Ca2(+)-ATPase activity and intracellular Ca2+ of HaCaT cells was decreased significantly. Terpene penetration enhancers perhaps changed the membrane fluidity and potentials of HaCaT cells by altering the Ca2+ balance of the cell inside and outside, resulting in the low skin permeability to increase the drug transdermal absorption.