ABSTRACT: The relationship between circulating fatty acid (FA) composition and childhood sleep disturbance remains largely unclear. We aimed to investigate the association of erythrocyte membrane FA composition with prevalence of sleep disturbance in Chinese children and adolescents. A cross-sectional survey was conducted among 2337 school-aged children and adolescents who completed a clinical assessment in Beijing, China. Presence of sleep disturbance was self-reported or parent-reported by questionnaires. Erythrocyte FAs were measured by gas chromatography, and desaturase activities were estimated by FA ratios. Multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for sleep disturbance across FA quartiles were calculated by a logistical regression model. We found higher proportions of erythrocyte phospholipid 24:0, 24:1n-9, and lower proportions of total n-3 polyunsaturated FA (PUFA), 22:5n-3 and 22:6n-3 in participants with sleep disturbance compared with those without. In the logistical regression models, significant inverse associations were found for total n-3 PUFA, 22:5n-3 and 22:6n-3, the highest versus lowest quartile ORs and 95% CIs were 0.57 (0.40, 0.82), 0.67 (0.47, 0.97) and 0.69 (0.49, 0.96), respectively. For per 1 SD difference of proportion, OR and 95% CI of prevalence of sleep disturbance was 0.91 (0.86, 0.97) for total n-3 PUFA, 0.90 (0.82, 0.98) for 22:5n-3, and 0.92 (0.86, 0.99) for 22:6n-3, respectively. No significant association was found for saturated fatty acids, monounsaturated fatty acids, n-6 polyunsaturated fatty acids or FA ratios. The present study suggested that erythrocyte n-3 PUFAs, especially 22:5n-3 and 22:6n-3, are inversely associated with prevalence of sleep disturbance in Chinese children and adolescents.