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Highlights • Left-behind children in China offers "natural experiments" of maternal separation. • Parent-child separation in early childhood associated with shorter telomere length. • Shortened TL partially explained adverse effects of separation on psychopathology. • Shortened TL could also be a consequence of psychopathology associated with separation. Abstract Background Given the ethical limitations of exposing children to experimentally manipulated adverse experiences, evidence of the effects of parent-child separation on subsequent psychopathology are based mostly on animal studies. Left-behind children phenomenon resulting from rural-urban mobility in China offers unique "natural experiments" to explore the long-term physical and mental health consequences of parent-child separation in childhood. Objective To test the associations between parent-child separation with telomere length (TL) and psychopathology during adolescence. Method A total of 710 adolescents (age: M = 16.86, SD = 1.52) were recruited from local schools in rural area of Fuyang, one of the top inland areas for outward migration in Anhui province, China. Parent-child separation was collected through face to face interview. The MacArthur Health & Behavior Questionnaire (HBQ) was used to assess internalizing and externalizing symptoms. Quantitative polymerase chain reaction was used to measure buccal TL. Results Nearly 60% (399/695) of the participants experienced separation from both parents. Childhood or persistent separation from parents was associated with increased internalizing symptoms (childhood: β = 0.13, 95% CI : 0.02, 0.23; persistent: β = 0.23, 95% CI : 0.14, 0.31), increased externalizing symptoms (childhood: β = 0.17, 95% CI : 0.03, 0.32; persistent: β = 0.23, 95% CI : 0.10, 0.35) and shorter telomere length (childhood: β = −0.16, 95% CI : −0.26, −0.05; persistent: β = −0.13, 95% CI : −0.22, −0.03). Shortened TL was estimated to explain 15.2% and 12.7% of the total effect of separation on internalizing and externalizing symptoms, while internalizing and externalizing symptoms explained 23.4% and 12.3% of the effect of separation on shortened TL. Conclusion Childhood and persistent parent-child separation, as experienced by rural left-behind children in China, associates with increased vulnerability for psychopathological symptoms and makers of cellular aging. The challenge for future research is to determine whether short telomere length is in fact a long-term consequence or an underlying vulnerability factor for future mental disorders. [ABSTRACT FROM AUTHOR]