Manganese (Mn) exposure can affect brain development. Whether Selenium (Se) can protect neonates against neurotoxicity from Mn exposure remains unclear. We investigated this issue in 933 mother-newborn pairs in Shanghai, China, from 2008 through 2009. Umbilical cord serum concentrations of Mn and Se were measured and Neonatal Behavioral Neurological Assessment (NBNA) tests were conducted. The scores <37 were defined as the low NBNA. The median concentrations of cord serum Mn and Se were 4.0 µg/L and 63.1 µg/L, respectively. After adjusting for potential confounders, the interaction between Se and Mn was observed. Cord blood Mn levels had different effects on NBNA scores stratified by different cord blood Se levels. With Se<P50 (<63.1 µg/L), Mn was negatively associated with NBNA scores (adjusted ß = -1.1, 95% CI: -1.3 to -0.9, p<0.001) and a higher cord blood Mn level increased the risk of low NBNA (adjusted OR = 5.7, 95% CI: 2.8 to 11.5, p<0.001). However, the adverse effect of Mn was reduced with Se ≥ P50 (≥ 63.1 µg/L) (NBNA: adjusted ß = 0.1, 95% CI: -0.3 to 0.5, p = 0.746; Low NBNA: adjusted OR = 4.5, 95% CI: 0.4 to 46.7, p = 0.205). Furthermore, the high Mn exposure group with a low Se level [Mn ≥ P75 (9.1 µg/L) and Se<P50 (63.1 µg/L)] had much lower NBNA scores than that of high Mn exposure group with a high Se level [Mn ≥ P75 (9.1 µg/L) and Se ≥ P50 (63.1 µg/L)] (38.0 ± 1.6 & 39.5 ± 0.9, p<0.001). Mn/Se ratio and NBNA scores were moderately correlated (r = -0.41, p<0.001). Our findings suggest that Se has a protective effect on neonates' brain development against neurotoxicity from prenatal exposure to Mn. Se supplementation should be considered during pregnancy, especially in areas with low natural Se.