Neuropeptide Y (NPY) is a major neural signaling molecule. NPY is produced by peripheral tissues, such as osteoblasts, and binds to the corresponding Y1 receptor that belongs to the G‑protein‑coupled receptor family. Osteoblast‑specific Y1 receptor knockout mice exhibit high bone mass, indicating a role of the NPY‑Y1 receptor axis in the regulation of bone homeostasis. In the bone microenvironment, peripheral nerve fibers and osteoblasts produce NPY. However, the effects of the Y1 receptor on osteoblasts remain unexplored. In the present study, an RNA interference approach was employed to target the Y1 receptor, in order to determine whether it may function to regulate the growth, differentiation and viability of osteoblasts. Knockdown of the Y1 receptor by small interfering RNA (siRNA) lead to induction of alkaline phosphatase (ALP) activity and mineralization in mouse MC3T3‑E1 osteoblast cells. In addition, the mRNA expression levels of ALP, osteocalcin, collagen (I) α1, and bone sialoprotein were significantly increased following transfection of a Y1 receptor siRNA. Furthermore, the mRNA expression levels of Runx2 and osterix were significantly increased; however, no significant alterations in cell proliferation and caspase‑3/7 activity were observed in Y1 receptor siRNA‑transfected cells when compared with non‑targeting controls. The results demonstrate that Y1 receptor inhibition may increase osteoblastic differentiation, which indicates a role of the Y1 receptor in the regulation of osteoblastic differentiation.