Glucose-regulated protein 78 (GRP78) is an important member of the heat shock protein 70 (HSP70) family, which plays important roles in the response to stimulation from various external environmental stimuli. In this study, the full-length cDNA of GRP78 (PmGRP78) was cloned from Penaeus monodon. The qRT-PCR analysis showed that PmGRP78 was ubiquitously expressed in all the tested shrimp tissues, and the expression pattern of PmGRP78 in the gill and hepatopancreas tissues after a pH challenge, osmotic stress, and heavy metal exposure was also detected. A chaperone activity assay was performed to study the function of PmGRP78, and the results showed that the recombinant PmGRP78 possesses biological activity and could protect alcohol dehydrogenase (ADH) from denaturing in vitro. To further explore the role played by PmGRP78 in vivo, RNA interference (RNAi) technology was adopted to silence the expression of PmGRP78 in P. monodon, and a cumulative mortality rate assay was carried out. In conclusion, we inferred that PmGRP78 may play important roles in the defence against environmental stresses in P. monodon.