Mechanical loading could affect bone remodeling, which involves the balance between bone resorption and formation. During bone remodeling, osteoblasts act as the primary sensors of mechanical signals, as well as the effectors to translate these signals into bone remodeling. Furthermore, osteoblasts express the Non-Neuronal Cholinergic System (NNCS), including acetylcholine (ACh) and α7 nicotinic Acetylcholine Receptor (α7nAChR), which regulates cellular function. However, the relationship between ACh/α7nAChR pathway and mechanical tension-induced bone remodeling remains unclear. Herein, we explored the effect of mechanical tension on osteoblasts, and the potential role of ACh/α7nAChR pathway in the tension-induced responses in osteoblasts. Specifically, MC3T3-E1 cells were subjected to a cyclic stretch in vitro using the Flexcell-5000™ Tension System. α7nAChR gene was knocked down with small interfering RNA (siRNA). Osteoblast proliferation, osteogenic function and the expression of the cholinergic system were assessed. According to the results, osteoblasts proliferation, osteogenesis-related factors expression [Runt-related Transcription Factor 2 (Runx2), Collagen Type-Ⅰ (Col1), Osteocalcin (Ocn), and Osteopontin (Opn)], and cholinergic system expression [acetylcholine (ACh), Carnitine Acetyltransferase (Carat), Vesicular Acetylcholine Transporter (Vacht), and α7 nicotinic Acetylcholine Receptor (α7nAChR)], these all increased initially, peaked at 8 h of tension, then declined with increasing tension time. Furthermore, mechanical tension with α7nAChR knocked down significantly decreased the early-stage osteogenesis-related genes and proteins expression of RUNX2 and COL1. In conclusion, mechanical tension exerted a time-dependent effect on osteoblasts proliferation, osteogenesis, and cholinergic system, which all increased initially, peaked at 8 h of tension, then declined with increasing tension duration. Furthermore, the ACh/α7nAChR pathway involved in early-stage osteogenesis induced by mechanical tension.