Exposure to neonicotinoid insecticides (NNIs) was proven to be harmful to organisms, however, there is a dearth of information regarding their occurrence and adverse effects on the general residents. Here, n = 120 human serum samples were collected from the same area of Wuxi city, Eastern China, and these serum samples were further analyzed for nine NNIs and nine target lipid molecules by use of ultrahigh performance liquid chromatography-quadrupole orbitrap high-resolution mass spectrometer. We observed that four out of nine NNIs exhibited relatively high detection frequencies (DF), and these NNIs were imidacloprid (IMI; DF = 28.3%), clothianidin (CLO; 16.7%), thiacloprid (THI; 14.2%), and acetamiprid (ACE; 12.5%), respectively, with 95th concentrations ranging from 32.0 to 427 pg/mL. Median concentrations of imidacloprid-equivalent total neonicotinoids (IMI) and ∑NNI were 46.6 pg/mL and 26 pg/mL, respectively. Five out of nine lipid molecules exhibited higher levels, that were docosahexaenoic acid [FA(22:6)], 18:0 phosphocholine [LysoPC(18:0)], 18:0 phosphoethanolamine [LysoPE(18:0)], D18:1-18:0 sphingomyelin [SM(d18:1/18:0)], and 18:1-18:1 diglycerol [DG(18:1/18:1)], respectively. More interestingly, we observed statistically significant correlations (student’s t-test, one-way ANOVA, or Mann-Whitney test; p < 0.05) between NNI levels and population characteristics (i.e. age, smoking, and health status). Beyond that, we also observed statistically significant correlations between levels of selected NNIs (CLO, ACE, or THI) and lipid molecules [LysoPE(18:0), SM(d18:1/18:0), and DG(18:1/18:1)]. Collectively, for the first time, we provided the information on contamination levels of NNIs in serum samples of general residents in China and demonstrated the associations between concentrations of NNIs and levels of lipid molecular species.