Updated project metadata. Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wildtype primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were enriched in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70RSK, and MTOR. Cell surface markers and matrisome proteins were also annotated.