Objective: Transcriptome sequencing and bioinformatics analysis were used to identify key genes and related pathways in osteosarcoma. Methods: osteosarcoma tissues and adjacent control tissues were collected, expression profiles were established by transcriptome microarray and differential expression genes (DEGs) were screened. The GO and KEGG enrichment analysis of DEGs was performed through online database. Results: A total of 5 pairs of osteosarcoma and para-tumor control tissues were included in this study. A total of 1211 genes with significantly up-regulated expression and 810 significantly down-regulated expression were screened in tumor tissues. GO analysis suggested that DEGs was mainly concentrated in biological processes such as tissue development, small molecule metabolism and cell cycle, while KEGG analysis suggested that DEGs was mainly involved in adhesion plaques, extracellular matrix receptor interactions, cancer signaling pathways and cell cycle related pathways. Conclusion: COL1A1, SPP1, POSTN, TYMS, TNC, ATP1A2, PDK4, CLIC5, ACTA1 and FABP4 may play an important role in the development of osteosarcoma.