p53, as a "Guardian of the Genome", plays an important role in cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis in different tissues including testis. p53 gene and its protein perform many essential roles for mammalian spermatogenesis. To explore its functions during spermatogenesis in Eriocheir sinensis, we have cloned and sequenced the cDNA (1,218 bp) of p53 from the testis by degenerating primer PCR and rapid-amplification of cDNA ends. The protein alignment of p53 shows the conserved DNA binding domain, dimerization site and zinc binding site consisted of the predicted structures. Phylogenetic analysis revealed that p53 was more closer to Marsupenaeus japonicus and Tigriopus japonicus than other examined species. Tissue expression analysis of p53 mRNA showed p53 was distinctly expressed in accessory sexual gland, muscle, gill, heart, hepatopancreas and testis. In situ hybridization revealed that the p53 mRNA was weakly distributed around the nucleus, but stronger in the invaginated acrosomal tubule at the early stage. At the middle stage, p53 mRNA signal was increased than the early stage and the signal displayed dot-like pattern on the surface of cup-like nucleus. The signal on acrosomal cap is stronger than on the acrosomal tubule, despite acrosomal tubule signal was also distinct. At the late stage, the signal was still mainly located in acrosomal cap and acrosomal tubule. Sporadic signal were found surrounding the cup-like nucleus, but they were very weak. In the mature sperm, the signal was dramatically decreased. Even though the signal on cup-like nucleus and acrosomal tubule were distinct, they were weaker than those in middle stage. Based on these results, we concluded that p53 may play an important role in formation of acrosome biogenesis and nuclear shaping during spermiogenesis of E. sinensis.