Introduction: Breast milk is a liquid secreted by the mother's breast glands and is useful for the baby. The World Health Organization (WHO) and the American Academy of Pediatrics (AAP) stated that the best alternative for breast milk is donor human milk (DHM). Until now, there is no donor HMB in Indonesia. This study aims to determine the relationship between knowledge and attitudes with the behavior of health workers regarding the acceptance of Human Milk Banks (HMB). Methods: An analytical cross-sectional study involved health workers at the pediatric ward of General Hospital Dr. Soetomo in 2020. The study involved 77 subjects consisting of doctors and nurses. Data was collected by questionnaires. A multiple linear regression test was used to determine the correlation between knowledge and attitudes with the behavior of health workers regarding the acceptance of HMB. Results: Among 77 research subjects, 42/77 subjects were doctors. 48/77 were female. 31/77 subjects were in the 25-29 years age group. 41/77 subjects were married. 38/77 subjects working period in the profession was 1-5 years. 67/77 subjects were Muslim. Knowledge was associated with the behavior of health workers (p=0.003), and attitudes were associated with the behavior of the health workers about the acceptance of the HMB (p=0.008), and there was a simultaneous correlation between knowledge and attitude with the behavior of health workers regarding the acceptance of HMB (p=0.000). Conclusion: These findings showed that knowledge and attitudes were associated with the behavior of health workers regarding the acceptance of HMB.