The human muscle-type nicotinic acetylcholine receptor α1β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. Graphical abstract created with http://biorender.com.