The ability of exogenous compounds to cross the blood-cerebrospinal fluid barrier (BCSFB) is a crucial factor in evaluating their potential neurotoxic risks. To date, limited research has reported the presence of parent neonicotinoids (p-NEOs) and metabolite NEOs (m-NEOs) in cerebrospinal fluid (CSF), not to mention their penetration efficiencies across BCSFB. Nine p-NEOs and 5 m-NEOs were measured in 159 paired serum and CSF samples. Seven p-NEOs and 5 m-NEOs were frequently detected in serum (median of 0.030-0.295 ng/mL), while 5 p-NEOs and 5 m-NEOs were commonly found in CSF (median of 0.032-0.283 ng/mL). Notably, concentrations of imidacloprid-olefin and acetamiprid-N-desmethyl exceeded those of imidacloprid (IMI) and acetamiprid (ACE), indicating that IMI and ACE predominantly existed as metabolites. The ratios (R CSF/serum) of NEO concentrations in CSF to those in paired serum were used to estimate penetration efficiencies across BCSFB. Their R CSF/serum (median of 0.468-0.992) were positively correlated with log P (r = 0.673, p = 0.033) while negatively correlated with water solubility (r = -0.745, p = 0.013), highlighting lipophilicity as a principal factor influencing BCSFB penetration. Additionally, R CSF/serum was significantly correlated with white blood cell count in CSF. This study provided a unique delineation of differential behaviors of p-NEOs and m-NEOs in crossing BCSFB, offering a novel perspective on their neurotoxic risks.