Meloidogyne incognita can infect multiple plant species. Proteins synthesized in the esophageal glands and secreted through the stylet of plant parasitic nematodes play critical roles in the plant-nematode interactions. Female M. incognita live for approximately 15days, embedded in a host plant, but their esophageal gland proteins have not yet been comprehensively analyzed. In this study, a new bacterium-contamination-resistant method for collecting soluble proteins from esophageal gland cells (SPEGC) of female M. incognita was established. Approximately 5μg of freeze-dried proteins could be extracted from 150 female M. incognita. Bands of a one-dimensional SDS-polyacrylamide gel were excised after electrophoresis of 20μg of protein and were analyzed. Two hundred and forty-six proteins from SPEGC of female M. incognita were identified by LC-MS/MS. Gene Ontology analysis suggests that many of the secreted proteins are involved in protein or carbohydrate metabolism and proteolysis. Some of the SPEGC (46.3%) were predicted to be secreted through classical or non-classical secretory pathways. The described method presents a new approach for the identification of proteins stored in SPEGC of an important plant parasitic nematode. This global proteomic profile of SPEGC provides a basis for future studies to elucidate the functions of proteins secreted from female M. incognita on plant responses.