Objective Among various causes of insomnia, stress is the most common and representative cause. Insomnia is also known to negatively affect the quality of life (QoL). The objective of this study was to explore the effect of stress on QoL and the mediating role of insomnia symptoms in the relationship between stress and QoL. Methods In this study, the mediating effect of insomnia symptoms on the relationship between stress and QoL was analyzed by enrolling 3,714 participants from the Ansung and Ansan cohorts of the Korea Association Resource project from 2001 to 2004. These cohort participants were asked about how much they felt stressed during their everyday life. Insomnia symptoms were evaluated by asking participants whether they had trouble sleeping such as difficulty in falling asleep, disrupted sleep, and early morning awakening due to the lack of a validated questionnaire for this cohort. QoL was evaluated using the World Health Organization QoL Scale Brief Version. Results In total, stress was positively associated with insomnia symptoms, which in turn predicted QoL. The same result could be derived from subgroup analysis according to sex, and it was confirmed that insomnia symptoms acted as a mediating factor more significantly in female than in male. Conclusion In this study, insomnia symptoms were confirmed to act as a significant mediating factor between stress and QoL, suggesting that insomnia symptoms should be actively identified and controlled to alleviate the negative effect of stress on QoL in clinical practice.