We investigated the impact of the human-specific protein CHRFAM7A (dupα7) on the function of α7 nicotinic acetylcholine receptors (nAChRs) in two different types of neurons: human induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. The CHRFAM7A co-assembles with CHRNA7 but lacks a major part of the extracellular N-terminal ligand-binding domain. We assessed the function of homomeric and hybrid pentameric receptors with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Both PNU282987 and choline bind to the orthosteric site of the α7 receptor. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists with the type-2 positive allosteric modulator (PAM) PNU120596. 4BP-TQS, on the other hand, activates receptors as an allosteric α7 ligand at a transmembrane binding site with minimal desensitization. The presence of CHRFAM7A had a major impact on α7 receptor function by significantly reducing calcium transients in response to all three agonists. Taken together, we demonstrate in primary human and mouse neurons that CHRFAM7A significantly inhibits α7 nAChRs.