Heat shock factors (HSFs) participate in the response to environmental stressors and regulate heat shock protein (Hsp) expression. This study describes the molecular characterization and expression of PmHSF1 in black tiger shrimp Penaeus monodon under heat stress. PmHSF1 expression was detected in several shrimp tissues: the highest in the lymphoid organ and the lowest in the eyestalk. Significant up-regulation of PmHSF1 expression was observed in hemocytes (p<0.05) following thermal stress. The expression of several PmHsps was rapidly induced following heat stress. Endogenous PmHSF1 protein was expressed in all three types of shrimp hemocyte and strongly induced under heat stress. The suppression of PmHSF1 expression by dsRNA-mediated gene silencing altered the expression of PmHsps, several antimicrobial genes, genes involved in the melanization process, and an antioxidant gene (PmSOD). PmHSF1 plays an important role in the thermal stress response, regulating the expression of Hsps and immune-related genes in P. monodon.