The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca2＋]c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe≤5.5) activated outwardly rectifying Cl− current (ICl,pH) with slow kinetics of voltage-dependent activation. ICl,pH was potently inhibited by an anion channel blocker 4,4`-diisothiocyanostilbene-2,2`-disulphonic acid (DIDS, 73.5% inhibition at 1ՌM). ICl,pH became more sensitive to pHe by raising temperature from 24oC to 37oC. HaCaT cells also expressed Ca2＋-activated Cl− current (ICl,Ca), and the amplitude of ICl,Ca was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca2＋] (⁘[Ca2＋]acid) of HaCaT cells. The ⁘[Ca2＋]acid was independent of extracellular Ca2＋, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ⁘[Ca2＋]acid. In summary, we found ICl,pH and ⁘[Ca2＋]acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.