OBJECTIVE: Women with Turner syndrome (TS) are at increased risk of reduced bone mineral density (BMD) due to primary ovarian insufficiency. This study aimed to assess longitudinal changes in BMD in TS patients receiving oral or transdermal hormone replacement therapy (HRT) and to identify factors associated with bone density decline. METHODS: This retrospective pilot study included 40 TS patients treated with oral or transdermal estradiol. The median age was 22 years in both the transdermal (20-30) and oral (20-27) groups. Median BMI was 23.1 (21.1-25.5) and 25.4 (22.1-29.1), respectively. Dual-energy X-ray absorptiometry (DEXA) of the lumbar spine and femoral neck was performed at baseline and follow-up. The primary outcome was the t-score at both sites. Univariable and multivariable logistic regression analyses were used to identify factors associated with declining t-scores. RESULTS: At baseline, 38.3% of patients had osteopenia, and 3.3% had osteoporosis. During follow-up, a decline in t-scores at at-least one skeletal site was observed in 47.5% of patients. No significant differences were found between oral and transdermal estradiol therapy regarding absolute t-scores, t-score changes, or the proportion of patients with declining t-scores (all p > 0.05). In multivariable analysis, lower vitamin D levels at follow-up were independently associated with declining t-scores. CONCLUSIONS: In TS patients, the route of estradiol administration did not significantly affect long-term BMD outcomes. Despite HRT, a substantial proportion of patients experienced bone loss. Vitamin D status has emerged as the most relevant modifiable factor associated with declining BMD, highlighting the need for bone health management beyond HRT.