Arabidopsis thaliana WRKY39, a transcription factor that is induced by heat stress, is a member of the group II WRKY proteins and responds to both abiotic and biotic stress. Heat-treated seeds and plants of WRKY39 knock-down mutants had increased susceptibility to heat stress, showing reduced germination, decreased survival, and elevated electrolyte leakage compared with wild-type plants. In contrast, WRKY39 over-expressing plants exhibited enhanced thermotolerance compared with wild-type plants. RT-PCR and qRT-PCR analysis of wrky39 mutants and WRKY39 over-expressing plants identified putative genes regulated by WRKY39. Consistent with a role for WRKY39 in heat tolerance, the expression levels of salicylic acid (SA)-regulated PR1 and SA-related MBF1c genes were down-regulated in wrky39 mutants. In contrast, over-expression of WRKY39 increased the expression of PR1 and MBF1c. The WRKY39 transcript was induced in response to treatment with SA or methyljasmonate. Analysis of heat stress-induced WRKY39 in defense signaling mutants, including coi1, ein2, and sid2, further indicated that WRKY39 was positively co-regulated by the SA and jasmonate (JA) signaling pathways. Together, these findings reveal that heat stress-induced WRKY39 positively regulates the cooperation between the SA- and JA-activated signaling pathways that mediate responses to heat stress.