The specialised DNA polymerase mu (pol mu) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol mu intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol mu(/) mouse embryonic fibroblasts (MEFs) we show that lack of pol mu results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol mu. Here we show pronounced senescence in pol mu(/) MEFs, associated with high levels of the tumor-suppressor p16(INK4A) and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol mu(/) MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol mu. Pol mu(/) MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.