Dysfunction of the endolymphatic sac (ES) is one of the etiologies of Meniere's disease (MD), the mechanism of which remains unclear. The aim of the present study was to explore the molecular pathological characteristics of ES during the development of MD. Metabolomic profiling of ES luminal fluid from patients with MD and patients with acoustic neuroma (AN) was performed. Diluted ES luminal fluid (ELF) samples were obtained from 10 patients who underwent endolymphatic duct blockage for the treatment of intractable MD and from 6 patients who underwent translabyrinthine surgery for AN. ELF analysis was performed using liquid chromatography-mass spectrometry before the raw data were normalized and subjected to subsequent statistical analysis by MetaboAnalyst. Using thresholds of P ≤ 0.05 and variable important in projection > 1, a total of 111 differential metabolites were screened in the ELF, including 52 metabolites in negative mode and 59 in positive mode. Furthermore, 15 differentially altered metabolites corresponding to 15 compound names were identified using a Student's t-test, including 7 significant increased metabolites and 8 significant decreased metabolites. Moreover, two differentially altered metabolites, hyaluronic acid (HA) and 4-hydroxynonenal (4-HNE), were validated to be upregulated in the epithelial lining of the ES, as well as in the subepithelial connective-tissue in patients with MD comparing with that in patients with AN. Among these differentially altered metabolites, an upregulated expression of HA detected in the ES lumen of the patients with MD was supposed to be associated with the increased endolymph in ES, while an increased level of 4-HNE found in the ELF of the patients with MD provided direct evidence to support that oxidative damage and inflammatory lesions underlie the mechanism of MD. Furthermore, citrate and ethylenediaminetetraacetic acid were detected to be decreased substantially in the ELF of the patients with MD, suggesting the elevated endolymphatic Ca2+ in the ears with chronic endolymphatic hydrops is likely to be associated with the reduction of these two chelators of Ca2+ in ES. The results in the present study indicate metabolomic analysis in the ELF of the patients with MD can potentially improve our understanding on the molecular pathophysiological mechanism in the ES during the development of MD.