Propofol is an intravenous general anesthetic that alters neuronal excitability by modulating agonist responses of pentameric ligand-gated ion channels (pLGICs). Evidence suggests that propofol enhancement of anion-selective pLGICs is mediated by a binding site between adjacent subunits, whereas propofol inhibition of cation-selective pLGICs occurs via a binding site contained within helices M1–M4 of individual subunits. We considered this idea by testing propofol modulation of homomeric human glycine receptors (GlyRs) and nematode glutamate-gated chloride channels (GluCls) recombinantly expressed in Xenopus laevis oocytes with electrophysiology. The Haemonchus contortus AVR-14B GluCl was inhibited by propofol with an IC₅₀ value of 252 ± 48 μm, providing the first example of propofol inhibition of an anion-selective pLGIC. Remarkably, inhibition was converted to enhancement by a single I18′S substitution in the channel-forming M2 helix (EC₅₀ = 979 ± 88 μm). When a previously identified site between adjacent subunits was disrupted by the M3 G329I substitution, both propofol inhibition and enhancement of GluCls were severely impaired (IC₅₀ and EC₅₀ values could not be calculated). Similarly, when the equivalent positions were examined in GlyRs, the M2 S18′I substitution significantly altered the maximum level of enhancement by propofol, and the M3 A288I substitution abolished propofol enhancement. These data are not consistent with separate binding sites for the opposing effects of propofol. Instead, these data suggest that propofol enhancement and inhibition are mediated by binding to a single site in anion-selective pLGICs, and the modulatory effect on channel gating depends on the M2 18′ residue.