Aims: Rat sarcoma virus (RAS)-induced tumorigenesis has been suggested to follow a three-stage model consisting of an initial RAS activation, senescence induction, and evasion of p53-dependent senescence checkpoints. While reactive oxygen species act as second messengers in RAS-induced senescence, they are also involved in oncogenic transformation by inducing proliferation and promoting mutations. In the current work, we investigated the role of extracellular superoxide dismutase (SOD3) in RAS-induced senescence and immortalization in vitro and in vivo. We used a mouse embryonic fibroblast (MEF) primary cell model along with immortalized and transformed human cell lines derived from papillary and anaplastic thyroid cancer. Results: Based on our data, sod3 RNA interference in H-RasV12-transduced cells markedly inhibited cell growth, while sod3 over-expression in MEFs initially caused a proliferative burst followed by the activation of DNA damage checkpoints, induction of p53-p21 signal transduction, and senescence. Subsequently, sod3-transduced MEF cells developed co-operative p21-p16 down-regulation and acquired transformed cell characteristics such as increased telomerase activity, loss of contact inhibition, growth in low-nutrient conditions, and in vivo tumorigenesis. Interestingly, as previously reported with RAS, we showed a dose-dependent response to SOD3 in vitro and in vivo involving transcriptional and non-transcriptional regulatory mechanisms. Innovation: SOD3 may mediate H-RasV12-induced initiation of primary cell immortalization. Conclusions: Our results indicate that SOD3 influences growth signaling in primary and cancer cells downstream of the ras oncogene and could serve as a therapy target at an early tumorigenesis phase.