In crustaceans, some of fundamental regulatory processes related to a range of physiological functions, including ovarian maturation, molting, glucose homeostasis, osmoregulation, etc., occur in the organs of the eyestalk. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites (X-organ/sinus gland, XO/SG) within the eyestalk. As unilateral eyestalk ablation was the most common method used to artificially induce ovarian maturation for farmed Litopenaeus vannamei, to better understand the reproductive regulation mechanism in L. vannamei, we have investigated the transcriptomes of the eyestalk during five ovary developmental stages with or without eyestalk ablation by high-throughput Illumina sequencing technology. The raw reads were assembled and clustered into 127,031 unigenes. Meanwhile, the differentially expressed genes (DEGs) between ovarian development stages were identified. We examined, through DEG enrichment analysis, eyestalk gene expression patterns for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, comparing natural to artificially induced ovarian maturation. We also identified a variety of transcripts that appear to be differentially expressed throughout ovarian maturation. These include transcripts that encode G-protein coupled receptors (GPCRs) and neuropeptides, such as the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and crustacean female sex hormone (CFSH). Furthermore, numerous exoskeleton formation-related genes were found to be down-regulated during ovarian maturation, including cuticle-like proteins, eclosion hormone (EH), and gastrolith-like proteins, of which the latter are the first reported in L. vannamei. Our work is the first reproduction-related investigation of L. vannamei focusing on the eyestalk at the whole transcriptome level. These findings provide novel insight into the function of the eyestalk in reproduction regulation.