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Background: Biopharmaceutical medicines have transformed the treatment of various long-term diseases, despite their high cost and limited availability. Due to their cost saving potentials, biosimilar medicines represent a new wave of therapy for several diseases in the next few years. Thus, pharmacists are uniquely placed to promote and enhance their uptake.Objectives: The objectives of this study were to assess Nigerian pharmacists' biosimilar medicine knowledge, attitudes, and practices.Methods: This was a cross-sectional, national online survey of conveniently sampled pharmacists in Nigeria. Data were collected with a validated 31-item biosimilar medicine knowledge, attitude, and practice questionnaire. The Pearson correlation (r) analysis was conducted to investigate the association among knowledge, attitude, and practice. A P value of < 0.05 was considered statistically significant.Results: Of the 600 pharmacists who were invited to participate in the survey, 411 completed the questionnaire giving a response rate of 68.5%. The mean knowledge score was 6.2 ± 3.0 out of a maximum score of 14. Most of the participants (n = 268, 65.2%) had overall knowledge scores of 1 to 7. The mean attitude score was 35.0 ± 8.8 out of a maximum score of 55, whereas that of practice was 18.7 ± 5.3 out of a maximum score of 30. Knowledge was significantly positively correlated with practice (r = 0.360).Conclusion: Most of the surveyed pharmacists had poor knowledge of biosimilar medicines, while their reported fair attitude did not translate to good practice. [ABSTRACT FROM AUTHOR]