CONTEXT: Maternal lipid levels affect birthweight and the long-term health of the offsprings. However, this association could be influenced by genetic and other common factors. OBJECTIVE: This work aimed to explore the relationship between maternal lipid levels and birthweight of two pregnancies in the same mother. METHODS: In this population-based cohort study, 705 women and their 1 410 offsprings were included. From an initial sample of women with more than one singleton birth in the database, we made the following exclusions: missing data for pre-pregnancy BMI, pregnancy weight gain, birthweight and lipid values; maternal age less than 19 or older than 44 years old; gestational age < 37 weeks or > 41weeks, gestational diabetes mellitus/diabetic. In the second and third trimesters, serum samples were collected for the determination of fasting total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels. Then we assessed the association between maternal lipids and birthweight. RESULTS: Infants of women whose 2nd-trimester TC increased by 10th-20th percentile (-0.92~-0.56 mmol/L) from 1st to 2nd pregnancy were 239.69 (62.32~417.06) g lighter at birth than were infants of women those of 40th-50th percentile (-0.20~-0.03 mmol/L). Parity, gestational age, neonatal gender, maternal pre-pregnancy body mass index, maternal weight gain, and 3rd-trimester TC and HDL-C were all associated with higher birth weight. Every unit increase in TC in the third trimester increases birthweight by 53.13 (14.32 ~91.94) g. CONCLUSION: Maternal TC level is associated with birthweight independent of shared genes. TC may be used to guide diet and predict birthweight combined with ultrasound and other indicators.