Background: Post-traumatic stress disorder (PTSD) is a psychiatric condition that may occur after exposure to traumatic experiences. The single prolonged stress (SPS) protocol, well replicated in rats, strongly affects conditioned fear responses, a key component of PTSD symptomatology. However, it has not yet been fully validated in mice. Objective: To validate an SPS model in mice in an extended contextual fear conditioned paradigm Methods: Adult male and female C57/B6 mice were subjected to four consecutive stressors (single session) followed by one week of recovery. Fear conditioning consisted of three footshocks (0.3 mA, 1 s, 1 min apart after 3 min of exploration). Extinction and spontaneous recovery involved 8 minutes of chamber exposure. Freezing was defined as the absence of movement except for respiration. Results: A sex-dependent difference was found during extinction. We observed increased freezing in SPS-exposed males on extinction day 2, while females showed the opposite effect during the mid-phase of extinction day 1. No effects observed during spontaneous recovery. Conclusion: Preliminary findings suggest that this SPS model for mice partially reproduces contextual fear conditioning effects observed in other rodent models. Further studies will include reinstatement of contextual memories and cued fear conditioning, to continue assessing the validity of SPS in mice as a model for PTSD research, which could add robustness by extending its use across species.