The present study aimed to investigate whether the cyclic stretch‑induced proliferation of human bladder smooth muscle cells (HBSMCs) is mediated by muscarinic (M) receptors, together with the signal transduction mechanisms involved in this process. HBSMCs seeded onto silicone membranes were subjected to different cyclic stretches (5, 10, 15 and 20%) for 6 and 12 h. As the effect of cyclic stretch on M2 and M3 mRNA expression levels was maximal at 6 h 10% stretch, all subsequent experiments were performed at this stretch. Western blot analysis was used to quantify M2, M3, protein kinase C (PKC) and phosphorylated (p)‑PKC protein expression levels, flow cytometry was employed to examine cell cycle distribution and a 5-bromo‑2-deoxyuridine (BrdU) incorporation assay was used to assess cell proliferation at this stretch. Subsequently, HBSMCs were exposed to different acetylcholine concentrations and/or cyclic stretch, M receptor antagonists [AF-DX16, an M2 receptor antagonist; 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), an M3 receptor antagonist and atropine, a non‑selective antagonist] and GF 109203X, a PKC antagonist, to assess the possible underlying signaling mechanisms. Cyclic stretch was found to increase the proliferation of HBSMCs and the expression levels of M2, M3, PKC and p‑PKC proteins. M receptor and PKC antagonists exerted no apparent effect on nonstretched cells, but reduced the incorporation of BrdU into stretched cells; the most pronounced effects were observed when non‑selective M receptor and PKC antagonists were applied. Notably, 4‑DAMP did not inhibit stretch‑induced PKC activation. These results indicate that the activation of the M3 receptor signaling pathway in stretch‑induced HBSMC proliferation occurs via PKC-independent mechanisms.