Understanding the protein composition of the follicular fluid from brown brocket deer would aid the development of a medium for in vitro embryo production, which would in turn contribute toward programs for species preservation. Proteomic shotgun is a sensitive tool for analysis of complex protein mixtures. Thus, this study aimed to use shotgun technique to investigate the proteome of fluid from small/medium and large follicles from brocket brown deer. The fluid was obtained by laparoscopy-guided follicular puncture from five females. Quantitative proteomic analysis was performed by multidimensional liquid chromatography coupled with mass spectrometry. A total of 226 proteins were identified in the follicular fluid, 53 and 60 of which were found only in small/medium and large follicles, respectively. One hundred and thirteen proteins were common to both groups of follicles. Quantitative analysis showed no significant differences (P > 0.05) in protein abundances in the follicular fluid from small/medium and large follicles. The proteins identified were classified by gene ontology terms, in silico interaction and assigned to 12 pathways indicated that proteins are involved in protein binding, catalytic activity, regulation of biological processes, extracellular matrix organization, and complement and coagulation cascades. In conclusion, these data add knowledge on the follicular development and provide original information on the follicular environment, which can contribute in the future to formulation of culture medium to use in embryo technology in brown brocket deer.