RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] and RASSF1C are two ubiquitously expressed isoforms of the RASSF1 gene. The promoter of RASSF1A is frequently hypermethylated, resulting in inactivation in various human cancers. RASSF1A is implicated in the regulation of apoptosis, microtubule stability and cell cycle arrest. However, little is known about the regulation and function of RASSF1C. In the present study we show that exogenously expressed RASSF1C is a very unstable protein that is highly polyubiquitylated and degraded via the proteasome. Furthermore, RASSF1C degradation is enhanced when cells are exposed to stress signals, such as UV irradiation. Mule, a HECT (homologous with E6-associated protein C-terminus) family E3 ligase, but not SCFβ-TrCP [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box and β-TrCP is β-bransducin repeat-containing protein] or CUL4 (cullin 4)-DDB1 (damage-specific DNA-binding protein 1), is the E3 ligase for RASSF1C under normal conditions, whereas both Mule and SCFβ-TrCP target RASSF1C degradation in response to UV irradiation. GSK3 (glycogen synthase kinase 3) phosphorylates RASSF1C to promote RASSF1C degradation subsequently, which is negatively regulated by the PI3K (phosphoinositide 3-kinase)/Akt pathway. Thus the present study reveals a novel regulation of RASSF1C and the potentially important role of RASSF1C in DNA damage responses.