Introduction: The cognitive effects of cross-sex hormone therapy (CSHT) are not well understood. In cisgender individuals, sex hormone therapy can impact neurotransmitter levels and structural anatomy. Similarly, in gender-diverse persons, CSHT has been associated with neural adaptations, such as growth in brain structures resembling those observed in cisgender individuals of the same sex. Hormone-related changes in learning and memory, as seen in menopause, are associated with physiological hypogonadism or a decline in hormones, such as estradiol. The present study examined the effect of estradiol administration in humans on glutamate concentration in brain regions involved in semantic and working memory (i.e., the dorsolateral prefrontal cortex (DLPFC), the posterior hippocampus, and the pregenual anterior cingulate cortex (ACC)) and its relationship with memory. Methods: Eighteen trans women (male biological sex assigned at birth) ceased CSHT for 30-days for a washout phase (t1) upon study enrollment to reach a hypogonadal state. Working and semantic memory, cognition, hormonal assays, and brain imaging were assessed. Participants resumed CSHT for 60-days for a replacement phase (t2), after which the same evaluations from t1 were repeated. Results: Estradiol increased among trans women after 60-days of resumed CSHT with significant improvements in semantic memory compared to the hypogonadal phase. Working memory recall was significantly and positively correlated to glutamate in the DLPFC during the reinstatement phase, although the relationship was not moderated by levels of estradiol. Discussion: These results may have clinical implications for the therapeutic effects of estradiol replacement serving as a protective factor against cognitive decline and impairment for trans women post-gonadectomy.