The nuclear receptor Tlx is a key regulator of embryonic and adult hippocampal neurogenesis and has been genetically linked to bipolar disorder. Mice lacking Tlx (Nr2e1−/−) display deficits in adult hippocampal neurogenesis and behavioural abnormalities. However, whether Tlx regulates behaviour during adolescence or in a sex-dependent manner remains unexplored. Therefore, we investigated the role of Tlx in a series of behavioural tasks in adolescent male and female mice with a spontaneous deletion of Tlx (Nr2e1−/− mice). Testing commenced at adolescence (postnatal day 28) and continued until adulthood (postnatal day 67). Adolescent male and female Nr2e1−/− mice were hyperactive in an open field, an effect that persisted in adulthood. Male but not female Nr2e1−/− mice exhibited reduced thigmotaxis during adolescence and adulthood. Impairments in rotarod motor performance developed in male and female Nr2e1−/− mice at the onset of adulthood. Spontaneous alternation in the Y-maze, a hippocampus-dependent task, was impaired in adolescent but not adult male and female Nr2e1−/− mice. Contextual fear conditioning was impaired in adolescent male Nr2e1−/− mice only, but both male and female adolescent Nr2e1−/− mice showed impaired cued fear conditioning, a hippocampal-amygdala dependent cognitive process. These deficits persisted into adulthood in males but not females. In conclusion, deletion of Tlx impairs motor, cognitive and anxiety-related behaviours during adolescence and adulthood in male and female mice with most effects occurring during adolescence rather than adulthood, independent of housing conditions. This suggests that Tlx has functions beyond regulation of adult hippocampal neurogenesis, and may be an important target in understanding neurobiological disorders.