5-HT3 receptors are members of the family of pentameric ligand gated ion channels (pLGICs). Each subunit has four transmembrane α-helices (M1-M4), with M4 being most distant from the central pore. Residues in this α-helix interact with adjacent lipids and the neighboring M1 and M3 helices, contributing to both receptor assembly and channel function. This study probes the role of each M4 receptor residue in the 5-HT3A receptor using mutagenesis and subsequent expression in HEK293 cells, probing functional parameters using fluorescence membrane potential sensitive dye. The data show that only one residue in M4 (Y441) and two flanking residues (D434 and W459) result in nonfunctional receptors when substituted with Ala: D434A and W459A-containing receptors ablate expression, while Y441A-containing receptor do not, suggesting the latter is involved in channel gating. Most other altered residues have wild-type-like properties, which is inconsistent with data from other pLGICs. Substitution of Y441 and W459 with other aromatics restores function, suggesting the π ring is important. Further substitutions indicate interactions of Y441 with D238 in M1, W459 with F144 in the Cys loop, and D434 with R251 in M2, data consistent with recently published structures. These regions are critical for transducing binding into gating, and thus interactions of these residues can explain their importance in the function of the 5-HT3 receptor. We also conclude that the small number of critical M4 residues compared to related receptors supports the hypothesis that M4 does not behave identically in all pLGICs.