In order to elucidate the functional and molecular mechanisms employed during accessory sex gland seasonal development, we performed suppression subtractive hybridization (SSH) experiments in the crab, Eriocheir sinensis, by constructing forward and reverse cDNA libraries enriched with up- or down-regulated transcript genes. A total of 175 ESTs were obtained from 180 randomly picked clones in the two libraries, with an average insert size of 450 bp, and a cloning efficiency > 90%. Comparative sequence analysis of ESTs with sequences reported in public genomic databases identified 88 unigenes, 35 of which were homologous to reported proteins, with the remainder unidentified. Unigene Annotation identified some unigenes via the Gene Ontology classification. Some of these unigenes had greater activity in the early stage of gland development, with an inferred function in cellular processes (involved in cell proliferation and differentiation), and the other genes with inferred functions in protein synthesis, energy metabolism, and signal transduction were more prevalent during the peak stage of gland development. Hence, genes elevated during the early stage may play roles in the process of rapid cell growth, while those elevated during the peak stage may play important roles in protein synthesis and secretion, as well as sperm capacitation and fertilization. SSH results were validated by the real-time PCR analysis of five candidate genes, whose expression correlated well with the SSH results, and exhibited relative expression levels that were five fold greater than those observed in the SSH libraries, confirming the SSH success.