Human acid-sensing ion channel 1b (hASIC1b) is a H⁺-gated amiloride -sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker, psalmotoxin-1, decrease the migration and proliferation of glioma cells. Protein kinase C (PKC) also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's C-terminus has been shown to interact with protein interacting with C kinase 1 (PICK1), which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine (A) to prevent phosphorylation, and to glutamic acid (E) or aspartic acid (D), to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild-type (WT), while S40E, S499A, and S499D currents were decreased. The PKC activators PMA and PdBu inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A, but had no effect on S499A or on S40A/S499A. The PKC activators or inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information which may help in designing potentially beneficial therapies in their treatment.