Background: In present study we performed whole transcriptome analysis of bone samples of 18 osteosarcoma patients confirmed with histology and we compared the result of tumoral to non-tumoral samples from the same patient. We sequenced total RNA from 36 samples (18 tumoral bone samples and 18 non-tumoral bone samples) matching in pairs for each osteosarcoma patient. Methods: We extracted RNA from 18 tumor normal paired osteosarcoma fresh bone samples. RNAseq data were analyzed with DESeq2 and Reactome packages of R. Results: We found 6775 genes expressed differentially between the normal bone and osteosarcoma tissues. The differential expression had FDR corrected statistical significance below 0.1. 4092 genes were up-regulated and 2683 were down-regulated. Among those genes, BTNL9, MMP14, ABCA10, ACACB, COL11A1 and PKM2 were expressed differentially with the highest significance between tumor and normal bone. Functional annotation with the Reactome identified significant changes in the pathways related to the extracellular matrix degradation and collagen biosynthesis. Activation of PKM2 indicates the Warburg effect in tumor tissues. Conclusions: We identified differential expression of 6775 genes between the normal and osteosarcoma tissues with very high confidence. The up-regulation of COL family genes (COL11A1, COL2A1, COL10A1, COL3A1, COL6A1) with interaction with some others indicates the changes in the regulation of the synthesis and degradation of collagen and extracellular matrix that seems to be an important mechanism of osteosarcoma