BackgroundThe family with sequence similarity 20-member C (FAM20C) kinase, a Golgi casein kinase, which is responsible for phosphorylating the majority of the extracellular phosphoproteins within S-x-E/pS motifs, and is fundamentally associated with multiple biological processes to maintain cell proliferation, biomineralization, migration, adhesion, and phosphate homeostasis. In dissecting how FAM20C regulates downstream molecules and potential mechanisms, however, there are multiple target molecules of FAM20C, particularly many phenomena remain elusive, such as changes in cell-autonomous behaviors, incompatibility in genotypes and phenotypes, and others.MethodsHere, assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), proteomics, and phosphoproteomics were performed inFam20c-dificient osteoblasts and to facilitate an integrated analysis and determine the impact of chromatin accessibility, genomic expression, protein alterations, signaling pathway, and post translational modifcations.ResultsBy combining ATAC-seq and RNA-seq, we identified TCF4 and Wnt signaling pathway as the key regulators inFam20c-dificient cells. Further, we showed Calpastatin/Calpain proteolysis system as a novel target axis for FAM20C to regulate cell migration and F-actin cytoskeleton by integrated analysis of proteomics and phosphoproteomics. Furthermore, Calpastatin/Calpain proteolysis system could negatively regulate the Wnt signaling pathway.ConclusionThese observations implied thatFam20cknockout osteoblasts would cause cell homeostatic imbalance, involving changes in multiple signaling pathways in the conduction system.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04275-4.