Background: The effect of maternal copper (Cu) level on the risk of spontaneous preterm birth (SPB) remains debate. Therefore, we conducted a prospective nested case-control study in Shanxi Province to investigate the relationship between maternal serum Cu concentration and SPB risk, as well as the potential mediation effect of lipid metabolism. Method: From an overall cohort of 4229 women, 147 women affected by SPB at 20–36 gestational weeks (cases) and 381 women who delivered at ≥37 gestational weeks (controls) were included in our nested case-control study. Maternal blood samples were collected during 4–22 gestational weeks, and the concentrations of Cu, total cholesterol (TC), and triglycerides (TG) were measured. Information on maternal social demographic characteristics were collected using questionnaires. Unconditional logistic regression models were used to estimate the associations of Cu, TC or TG levels with SPB risk. Linear regressions were used to assess the relationships between concentrations of Cu and TC or TG. Results: Serum Cu concentrations in the case group (median: 184 μg/dL) were significantly higher than those in the control group (median: 166 μg/dL, p