BACKGROUND: This study aimed to perform a systematic review and meta-analysis of randomized controlled trials investigating the effect of exercise on high-density lipoprotein (HDL) levels in middle-aged and older individuals. METHODS: A systematic search was conducted in PubMed, Web of Science, and EMBASE, including meta-analyses of randomized controlled trials that investigated the effects of exercise on HDL-C levels in middle-aged and older individuals. RESULTS: Forty-two trials were analyzed. We found that exercise had a significant effect on HDL cholesterol levels (standard mean difference [95% confidence interval] = 0.22 [0.09-0.35], Z = 3.28, P = .001, I2 = 54%), and the heterogeneity across the studies was significant and high. After subgroup analysis, we found that aerobic exercise had the most significant effect on the levels of HDLs in middle-aged and older individuals, which was better than that of other types of exercise. CONCLUSIONS: Aerobic exercise significantly improves HDL levels in middle-aged and older individuals, but resistance and stretching exercises had no significant effect on HDL levels.