PURPOSE. Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl-) release through swelling-activated channels (I-Cl,I-Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl- (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. METHODS. Gene expression was studied with quantitative PCR, supplemented by reverse-transcriptase PCR andWestern immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. RESULTS. Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by I-Cl,I-Swell (-14 to -21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCC(inh-)A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, I-Cl,I-Swell and the regulatory volume response to hyposmotic swelling. CONCLUSIONS. Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, I-Cl,I-Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.