Exosomes are membrane vesicles of endocytic origin released by many cell types. The molecular composition of exosomes reflects the specialised functions of their original cells. For example, these vesicles can mediate communication through their ability to bind to target cells, facilitating processes such as vascular homeostasis and antigen presentation. Although the proteomes of exosomes from several cell types are known, exploration of exosomes from additional cell types may improve our understanding of their potential physiological roles. Here, we describe the isolation and characterisation of exosomes isolated from the culture medium of murine fibroblast NIH3T3 cells and Ras‐transformed NIH3T3 cells. The vesicular nature and size (30–100 nm) of the purified fibroblast exosomes was confirmed by electron microscopy. 2‐D difference gel electrophoresis (DIGE) was used to compare protein profiles of exosomes secreted from NIH3T3 cells and Ras‐transformed NIH3T3 cells. LC‐MS/MS sequencing identified proteins in 188 protein spots in the exosomes from the two cell lines, many of which have been previously identified in exosomes from other cell types. However, some proteins identified are novel for fibroblast exosomes, such as Serpin B6. Over 34 proteins, including milk fat globule EGF factor 8 (lactadherin), collagen α‐1 (VI), 14‐3‐3 isoforms, guanine nucleotide‐binding proteins (G proteins), the eukaryotic translation initiation factors elF‐3 γ and elF‐5A accumulated (>2‐fold) in exosomes upon Ras‐induced oncogenic transformation. Significantly, the 10.4‐fold increase in v‐Ha‐Ras p21 protein in exosomes derived from Ras‐transformed NIH3T3 cells suggests that exosome secretion may be implicated in eradication of obsolete proteins.