Background: Prior research has widely recognized that the most essential negative predictors in people with chronic diseases’ quality of life are insomnia, fatigue, and pain. To date, there are little to no studies that have identified how these physical symptoms perform together and influence the quality of life, especially among people living with Human Immunodeficiency Virus (HIV). This study aimed to examine the relationships between insomnia, fatigue, pain, and quality of life in people with HIV. Methods: People living with HIV (N = 200) who met the antiretroviral therapy criteria were recruited. Standardized scales for fatigue, pain, insomnia, antiretroviral therapy adherence, substance use, and quality of life were used to measure key study variables. Results: Results showed with 95% confidence intervals that the increasing levels of fatigue (B = 1.026, p < 0.001) and insomnia (B = –0.589, p = 0.003) were negatively associated with quality of life in people living with HIV. The effect of fatigue on quality of life was significantly mediated via insomnia (Sobel test of mediation effect = 0.225, z = 2.883, p = 0.004). The increasing levels of pain (B = –1.682, p < 0.001) and insomnia (B = –1.171, p < 0.001) were negatively associated with quality of life. Conclusions: The findings of moderated mediation analysis clarified the complex mechanism of how fatigue, insomnia, and opioid use influenced the quality of life and how pain, insomnia, and antiretroviral therapy adherence influence quality of life. It emphasizes the need for further interventions to target this complex interplay among physical symptoms and substance use to enhance the quality of life with antiretroviral therapy in people living with HIV. © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023.