The side effects of soft drinks are still an important challenge especially for the expressed genes of foetuses that regulate ossification. For that purpose, we investigate the expression of R-spending2 (RSPO2), Hydroxyacid Oxidase 1 (HAO1), and runt related transcription factor 2 (RUNX2) genes and the foetuses skeletal malformation due to maternal Pepsi consumption. Pregnant rats were divided into control group that was administrated orally with distilled water, group1 from day 1 to day 7 was orally administrated with 2.5 ml/day of Pepsi, group2 from day 1 to day 7 was administrated orally with 5ml/day of Pepsi, group 3 from day 8 to day 20 was orally administrated with 2.5 ml/day of Pepsi, and group 4 from day 8 to day 20 was orally administrated with 5 ml/day of Pepsi. Gene expression analysis revealed that the RSPO2 gene is significantly decreased with increasing the dosage of soft drinks during the 1st stage of pregnancy. Conversely, there is a significant increase in the HAO1 gene in 1st stage group relative to the control group. RUNX2 gene is significantly decreased in group1 and group 2 while it was significantly increased in groups 3 and 4 regarding the control group. Pepsi administration caused retarded body length and weight, ossification and lengths of some bones, and shortness of others. Different bones are seriously affected by Pepsi. Therefore, our findings reveal the effect of soft drink consumption on skeletal malformation and the RSPO2, HAO1, and RUNX2 genes that can be used as biomarkers for skeletal modulation.