Background: Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E. Methods: Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography. Results: In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk. Conclusion: The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk.