<b><i>Background:</i></b> Hormonal variations are known to influence the course of multiple sclerosis (MS). <b><i>Objectives:</i></b> We aimed to evaluate the impact of menopause in MS course, including disease activity and disability progression. <b><i>Methods:</i></b> We conducted a retrospective longitudinal cohort study including all women, older than 44, post-menopausal, with a diagnosis of MS at least 1 year before menopause. We evaluated the impact of menopause in MS course comparing clinical and radiologic outcomes within 5 years before and after menopause. We repeated the analysis in subgroups of patients without disease-modifying treatment (DMT) change or co-morbidities diagnosed during the observation period, considering that those factors might also impact MS outcomes. <b><i>Results:</i></b> Thirty-seven women, with a mean age at the time of menopause of 49.8 (±4.06) years were included in the analysis. Within 5 years following menopause, we observed a decrease in the annualized relapse rate (0.37 ± 0.35 pre-menopause vs. 0.08 ± 0.18 post-menopause, <i>p</i> &#x3c; 0.001) compared with the same period before menopause, while the EDSS progression rate remained stable (0.13 ± 0.24 EDSS point/year pre-menopausal vs. 0.13 ± 0.18 post-menopause, <i>p</i> = 0.935). EDSS progression events frequency was similar before and after the menopause (37.8 vs. 48.6%, respectively, <i>p</i> = 0.424). These observations persisted in patients’ subgroups without DMT switch or co-morbidities. <b><i>Conclusions:</i></b> Following menopause, we observed a reduction in the relapse rate, but the disability progression continued at a similar rate, compared to the pre-menopausal period. These observations persisted in the subgroup of patients without changes in DMT or co-morbidities diagnosed during the observation period.