SKT11, an important tumor suppressor, is a member of the serine/threonine kinase family and plays a crucial role in tumor invasion and metastasis by activated adenine monophosphate-activated protein kinase (AMPK) and AMPK-related kinase proteins. However, few studies have elaborated its regulations of development and metabolism of cartilage, as well as skeleton. This study was aimed to investigate the role of Stk11-knockout in chondrocyte by bioinformatics analysis. The gene expression profiles for Stk11-knockout and wild-type mice were downloaded from the Gene Expression Omnibus (GEO) database. A total of 1104 differentially expressed genes (DEGs) were identified by Affymetrix Expression Console and Transcriptome Analysis Console (TAC) software, including 560 up-regulated and 544 down-regulated genes. The protein-protein interaction (PPI) networks were built by mapping DEGs into STRING, in which hub genes such as Fos, Pdgfrb, Pdgfra, Flt1/Vegfr1, Smad3, Mapk14, Twist and Aurkb were further identified. For the up-regulated genes, PI3K-AKT signaling pathway and Wnt signaling pathway were two main pathways in the KEGG analysis, and ossification and extracellular matrix organization were involved in the Gene Ontology (GO) analysis. On the other hand, the down regulated genes were mainly involved in systemic lupus erythematosus and alcoholism pathways, and B cell receptor signaling pathway and immune system process biological processes. MiRNA-9, miRNA-134, miRNA-492, miRNA-224 and miRNA-142-5p were identified as key regulators in the miRNAs-DEG regulatory network. Additionally, OSF2/RUNX2, and NFAT regulated DEGs collectively in the transcription factor regulatory network. The results of RT-PCR verified that the expression of hub genes, transcription factors and miRNAs in our experiment were basically consistent with the microarray hybridization. In this study, we provide an insight into the role of Stk11 in chondrocyte and identify novel genes related to Stk11.