AimsIncreasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production. Main methodsIn the present study we analysed 16week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R–KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR. Key findingsA significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R–KO using Micro-CT analysis. Bending stiffness of M3R–KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression. SignificanceOur results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R–KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.