We investigated the impact of the human-specific protein CHRFAM7A (also referred to as “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. CHRFAM7A co-assembles with α7 (also referred to as CHRNA7) but lacks a major part of the extracellular N-terminal ligand-binding domain. We assessed the function of α7 in these preparations with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously we demonstrate in primary human iPS-derived and in mouse neurons that CHRFAM7A has a major impact on nicotinic α7 receptors by reducing calcium transients in response to all three agonists.