Osteoporosis (OP) is an aging-associated condition that significantly affects the quality of life in aging humans and has few treatment options. As an important marker, recombinant immunoglobulin superfamily containing leucine-rich repeat (ISLR) could be used as a gene therapy for OP. We herein used alkaline phosphatase (ALP) and alizarin red S (ARS) staining to evaluate osteogenic differentiation potential. Micro-computed tomography was used to determine bone formation and for image reconstruction of the trabecular distribution of the femur. ISLR knockout increased osteogenic differentiation and mineral deposition, consistent with the results in Ocn-cre:ISLRflox/flox mice and pCMV6-Islr-GFP (ISLR-overexpression, ISLR-OE) mice. ISLR negatively regulates osteogenic differentiation through the bone morphogenetic protein 4 (BMP4)-Smad-ColIα1/Ocn axis, promoting proteasomal degradation of BMP4. ISLR interacted with BMP4 to regulate osteogenic differentiation. Moreover, interference of ISLR expression was potentially therapeutic for bone loss treatment. These results demonstrated a new mechanism of osteogenic differentiation that could be used for developing effective bone therapies to treat OP.