In recent years, a disease outbreak occurred in >50 percent of Litopenaeus vannamei farms at about 30 days after frys are released into the ponds in southern China. About 80% culture shrimps were dead in culture period. The clinical symptoms of the diseased shrimps include atrophy and necrosis of the hepatopancreas, weakened activity, and shrimp found dead in the bottom of the pond. The farmers in China usually called this symptom as “hidden death disease”. Since hepatopancreas atrophy and necrosis are the main symptoms of the disease, so we called this disease as hepatopancreas necrosis syndrome (HPNS). In March 2012 to October 2013, we have collected the diseased L. vannamei with the symptoms of HPNS in 12 farms from Guangdong, Hainan and Guangxi provinces, China. Histopathology showed necrosis of hepatopancreas of the diseased shrimps, disappearance of some hepatopancreatic epidermal cells, and connective tissue. We used PCR to detect the presence of 11 viruses in 305 HPNS shrimps and performed artificial viral infection to healthy shrimps. The artificially infected shrimps did not show symptoms of HPNS. A total of 383 bacteria strains were identified from hepatopancreas, hemolymph and intestine of 63 diseased shrimps. These bacteria belong to 10 genera, 49 species. We isolated Vibrio parahaemolyticus from 38 shrimps, Bacillus cereus from 34 shrimps, B. thuringiensis from 20 shrimps and V. cholera from 19 shrimps. Injection and immersion of V. parahaemolyticus and B. thuringiensis can cause symptoms of HPNS. The results suggest that multiple bacteria were involved in HPNS of L. vannamei.