Chlorpyrifos is an organophosphate insecticide used primarily to control pests on a variety of food and feed crops. Humans are directly or indirectly exposed to this pesticide through food, air, and occupation. The ill effects of chlorpyrifos on various organ systems of human has been widely documented, but less is known about its influence on human bones. To analyze the effect of chlorpyrifos and its metabolites 3,5,6-trichloro-2-pyridinol (TCPy) on the skeletal system of the chick embryo. Fertilized chick eggs were exposed to different concentrations of chlorpyrifos and its metabolite 3,5,6-TCPy on 1.5 days of incubation. The proximal phalanx of 18-day-old embryos was analyzed for defects in growth and ossification through histopathology, immunohistochemistry, angiogenesis assay, and gene expression study. Dose-dependent variations in developing bone of chick embryo were observed. Histochemical and histomorphometry studies of proximal phalanx showed increased in the growth plate length (F(9, 59)=228.9509, p=.00001) with a reduction in the total length of the phalanx (F(9, 59)=109.9905, p=.00001), decreased mineralization (F(9, 59)=224.6872, p=.00001), decreased blood islands in the bone marrow (F(9, 59)=7.7083, p=.0001) of chlorpyrifos, and 3,5,6-TCPy-exposed group. Significant downregulations in the expression patterns of the transcription factors, such as SOX9, RUNX2, and ALP, were also observed. Chlorpyrifos and its metabolite 3,5,6-TCPy exposure alters the chondrogenesis in the growth plate cartilage of long bone in chick embryo. The pesticide and its metabolite also interfere in ossification.