Abstract Objective To assess the impact of the distance education course "Sickle Cell Disease: Primary Health Care Line" on knowledge acquisition of professional healthcare providers. Methods A cross-sectional study was conducted with a quantitative approach at the Educational and Support Center for Hemoglobinopathies (Cehmob-MG), state of Minas Gerais, Brazil, in 2016. One hundred and fifty-three out of 300 professional healthcare providers were invited to participate in the proposed distance course. Of the participating professional healthcare providers, 72 (47%) successfully concluded the course (Group A), whereas 81 (53%) did not complete their course assignments and did not meet the minimum requirements for regular attendance (Group B). Knowledge acquisition was assessed with the Knowledge of Sickle Cell Disease Instrument, DFConhecimento, applied using the web tool eSurv. Univariate analysis by Poisson regression was employed to assess the influence of sociodemographic variables on the DFConhecimento score and to select variables to compose the initial multivariate regression model (p-value < 0.20). The analysis was performed in the statistical programming environment R. Results The average score was 9.76 for Group A and 6.54 for Group B. The two groups were considered statistically different (p-value < 0.05) for all items with the proportion of correct items being greater in Group A. Professional healthcare providers who concluded the course had a significantly higher DFConhecimento score (45%) when compared to those who did not successfully conclude the course. Conclusion Participation in a distance education course on sickle cell disease had a positive impact on the acquisition of knowledge about the disease by professional healthcare providers.