ABSTRACT Although the association between self-regulation of fatigue and health-related quality of life (HRQoL) has been confirmed, the potential mechanism remains unclear. This study aimed to explore the role of health literacy, health behavior, and exercise frequency in the relationship among middle-aged and elderly patients with recurrent stroke. A cross-sectional survey was conducted. A total of 176 patients completed the survey, in which self-regulation of fatigue, HRQoL, health literacy and health behavior were measured by questionnaires. Based on Bootstrap analyses, a moderating sequential mediation model using PROCESS software was constructed with health literacy and health behavior as mediators and exercise frequency as the moderator. Of the participants, the mean age was 65.44 ± 12.43 years. Self-regulation of fatigue was found to affect HRQoL indirectly through two significant mediation pathways: (1) health literacy (β=-0.11, 95%CI = −0.20, −0.03), which accounted for 28.79% of the total effect, and (2) health literacy and health behavior (β=-0.02, 95%CI = −0.05, −0.00), which accounted for 4.80% of the total effect. Exercise frequency moderated the relationship between self-regulating fatigue and HRQoL. Specifically, the interaction term between self-regulating fatigue and exercise frequency significantly predicted HRQoL (β = 0. 25, t = 2.55, p < 0.05). These findings highlight the role of health literacy and health behavior as sequential mediators of the relationship between self-regulating fatigue and HRQoL. Moreover, exercise frequency moderated the relationship between self-regulating fatigue and HRQoL. Encouraging patients with recurrent stroke to increase exercise frequency appropriately might improve HRQoL for patients with poor health literacy and health behavior.