Healthcare-Acquired Infections (HAIs) often be an adverse effect of health service that increase the morbidity, mortality, and cost in hospitalized patients. One of the ways to prevent HAIs is using a standardized personal protective equipment (PPE). Purpose: To analyze the relations between knowledge and the adherence is using personal protective equipment (PPE) in hemodialysis employees at PKU Muhammadiyah Gamping Hospital to lessen the number of HAIs in Hemodialysis unit PKU Muhammadiyah Gamping Hospital. Methods: This is a quantitative with analytic-observational design and cross-sectional approach’s research. They are collected by a total sampling technique. Results and Discussion: 80% of all respondents have the highest level of knowledge while 7 respondents (70%) obey in using the PPE. There is a significant positive relation between knowledge and hemodialysis employees’ adherence in using PPE in Hemodialysis unit PKU Muhammadiyah Gamping Hospital with statistical calculation using Fisher’s Exact Test (p=0,022). The higher the knowledge about PPE, the higher the adherence in using PPE in Hemodialysis unit PKU Muhammadiyah Gamping Hospital (p=0,013) using Spearman’s correlation. Conclusion: This research concluded that there is a relation between knowledge and hemodialysis employees’ adherence in using PPE in Hemodialysis unit PKU Muhammadiyah Gamping Hospital. The higher the knowledge about PPE, the higher the adherence.