Nitric oxide (NO), mainly produced by neuronal nitric oxide synthase (nNOS) in the brain, has been implicated in stress responses and the pathophysiology of post-traumatic stress disorder (PTSD). Our group previously showed that inducible nitric oxide synthase (iNOS) knockout (KO) male mice exhibit compensatory changes in nNOS expression in the medial prefrontal cortex (mPFC) and impaired fear extinction, suggesting that this genetic model may be relevant for studying PTSD-related phenotypes. Given the frequent comorbidity of PTSD with anxiety and depression, and the marked underrepresentation of females in neuropsychopharmacology research, we performed a behavioral characterization of male and female iNOS KO mice, focusing on aversive memory, anxiety-, and depression-like behaviors. To our knowledge, this is the first systematic behavioral study of female iNOS KO mice, which is particularly relevant given that females are twice as likely to develop psychiatric disorders. We observed that female iNOS KO mice exhibited increased anxiety-like behavior in the elevated plus maze test (EPMT), whereas males showed antidepressant-like behavior in the forced swim test (FST). No general cognitive deficits were found in the Y-maze or object recognition (OR) tests in either sex. However, male iNOS KO mice exhibited deficits in fear extinction memory and extinction retrieval in both contextual and cued fear conditioning. These findings indicate that iNOS KO mice present sex-dependent behavioral phenotypes and may serve as a genetic model to investigate disorders related to fear memory, such as PTSD, and highlight the importance of considering sex as a biological variable in research.