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Introduction: Appointment spacing model (ASM) of care is crucial for human immunodeficiency viruses (HIV) patients receiving antiretroviral therapy in order to improve service quality and patient’s clinical outcomes, including viral suppression. However, there is a paucity of information about the effectiveness of ASM on viral suppression. Therefore, this study aimed to assess the level and trends of virological suppression and associated factors among patients on antiretroviral therapy enrolled into ASM in Northwest Ethiopia. Material and methods: An interrupted time series study design was conducted among 272 adult HIV-positive patients, who were stable and enrolled into ASM. They were selected by using a systematic random sampling technique. Data were collected from patients’ charts, registration books, and computer databases using abstraction sheets. Regression coefficients with a 95% confidence interval (CI) computed, and variables having less than p-value = 0.05 in the segmented regression model were considered significant predictors of virological suppression. Results: This study revealed that virological suppression was decreased from the baseline of 99.22% to 96% after the implementation of ASM. The trends of virological suppression were significantly decreased by 1.38 (95% CI: –2.2, –0.5%, p = 0.0007) after the implementation of ASM. Poor adherence was the most influential variable that caused level and trend decrements over time (p = 0.04). Conclusions: The level of virological suppression was significantly reduced after the implementation of ASM. The most significant factor associated with decreased levels and trends over time was poor adherence. It would be beneficial to assess and maintain good adherence of patients on antiretroviral therapy throughout the clinical visit during the implementation of ASM. [ABSTRACT FROM AUTHOR]