We performed 4D label free-based quantitative proteomic analysis of Eimeria tenella unsporulated oocysts and sporulated oocysts. Briefly, protein extraction was performed using liquid nitrogen grinding and sonication on ice in SDT buffer (4% SDS, 100 mM Tris-HCl, pH7.6). After trypsin digestion, the peptides were separated on Easy-nLC1000 liquid chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) with a trap column (Thermo Fisher Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) and analytical column (Thermo Fisher Scientific Easy Column, 25 cm long, 75 μm inner diameter, 1.9 μm resin). LC-MS/MS analysis was conducted on a timsTOF Pro mass spectrometry. The resulting LC-MS/MS data files were processed with the MaxQuant software (version 1.5.3.17) for identification and quantitation analysis.