In this study, the effects of the probiotic Lactobacillus rhamnosus IMC501, on oocyte maturational competence acquiring was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (IIIa) to 17,20-dihydroxy-4-pregnen-3-one (MIH) and their in vitro maturation. The acquiring of competence of the IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activinbetaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte maturational competence acquisition and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted from the same follicles and from the relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the GI tract and modulate the microbial communities causing a clear shift in them, specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophiles. At the same time PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicles development were also analyzed by FPA (Focal Plane Array) FT-IR Imaging, a technique which provides the overall biochemical composition of samples. Changes were found above all in IIIa follicles from probiotic exposed females; the modifications, observed in proteins secondary structures, as well as in hydration and in bands related to phosphate moieties, allow us to hypothesize that probiotics act at this follicle stage affecting maturation phase.