Neonatal sepsis is an inflammatory system syndrome and a main cause of neonatal mortality. However, there is a lack of ideal biomarkers for early neonatal sepsis diagnosis. The aim of this study was to evaluate the clinical significance of miR-141 in sepsis in neonates, and explore the regulatory effects of miR-141 on inflammation in monocytes. This study used qRT-PCR to calculate the expression of miR-141 in the serum of septic neonates. The diagnostic values of procalcitonin (PCT) and serum miR-141 were evaluated by receiver operating characteristic (ROC) curves. The relationship between miR-141 and TLR4 was determined using luciferase reporter assay. An inflammation model was established using monocytes with lipopolysaccharide (LPS) treatment. ELISA assay was used to analyze the levels of pro-inflammatory cytokines. The expression of miR-141 in neonatal sepsis was significantly lower than healthy controls. ROC curves showed that miR-141 had diagnostic accuracy. LPS stimulation in monocytes led to a decrease in the expression of miR-141. A luciferase reporter assay proved that miR-141 targeted TLR4, and a negative correlation of miR-141 with TLR4 was found in septic neonates. ELISA results demonstrated that the overexpression of miR-141 inhibited LPS-induced inflammation in monocytes. In conclusion, serum decreased miR-141 expression served as a candidate diagnostic biomarker of neonatal sepsis. TLR4 is a target gene of miR-141, which may mediate the inhibitory effects of miR-141 overexpression on LPS-induced inflammation in monocytes. Therefore, miR-141 is expected to be a potential diagnostic biomarker and a therapeutic target in neonatal sepsis.