Female ovarian development is crucial for the successful propagation of sexually reproducing organisms that involve several physiological events, including vitellogenin synthesis, oocyte proliferation, and metabolism. These processes are controlled by several genes. Among them, a leucine-tyrosine-arginine motif-containing protein 5 (LYRM5) was found to be differentially expressed between undeveloped and mature ovaries, in the transcriptome of Fenneropenaeus merguiensis's ovaries. This study aimed to investigate the expression analysis and the role of LYRM5 in vitellogenesis of female banana shrimp, F. merguiensis. The FmLYRM5 was phylogenetically analyzed to be a member of LYRM5 and showed high similarity to LYRM5 of other species. The FmLYRM5 was highly expressed in eyestalks, thoracic ganglia, and hepatopancreas, and less in brain, gills, and ovary. Its expression was significantly affected in the ovaries by the developmental stage and was decreased in the matured shrimp, but was unchanged in hepatopancreas. Also, it was up-regulated by 20-hydroxyecdysone (20-HE), methyl farnesoate (MF) and serotonin (5-HT) treatments in the primary ovarian cells. The FmLYRM5 localizes at the mitochondrial compartment in the CHO cells. The overexpression of FmLYRM5 in female shrimp, induced by the recombinant plasmid (pLYRM5His) injection, showed increased vitellogenin expression and oocyte proliferation. Furthermore, the total sugar level in hemolymph (1526.73 ± 392.05 μg/ml) and ovary (1.53 ± 0.3 μg/mg ovary) was induced in shrimp injected with pLYRM5His on day 14 compared with that in hemolymph (139.10 ± 17.5 μg/ml) and ovary (0.71 ± 0.3 μg/mg ovary) of control shrimp. The total lipid level was increased only in the hemolymph of FmLYRM5-overexpressing shrimp (54.19 ± 23.1 μg/ml) compared with that of control shrimp (5.52 ± 1.4 μg/ml) but was unchanged in their ovaries. Our study indicates one mitochondrial factor that plays roles in stimulating vitellogenesis and in metabolic regulation during ovarian development.