It is known that the polyamine (PA) biosynthetic pathway is modulated at the transcriptional level during abiotic stresses. Here we studied the expression of PA biosynthetic pathway genes upon exposure to heat shock (HS) in Arabidopsis and showed that the spermine (Spm) synthase gene (SPMS) and S-adenosylmethionine decarboxylase 2 gene are induced at the earliest stage, followed by the induction of the arginine decarboxylase 2 gene. Correspondingly, Spm content increased linearly upon HS, and putrescine (Put) and spermidine (Spd) content also increased but not thermospermine (T-Spm) content. Exogenously applied Spm had a potential to protect Arabidopsis plants from HS-induced damage. Such protection was also observed to the same extent with T-Spm and by Spd to a lesser extent but not by Put. Then we tested whether altered endogenous Spm content affects sensitivity to HS using both transgenic plants overexpressing SPMS and a Spm deficient (spms) mutant plant. The result revealed that the higher the Spm content the higher the thermotolerance. Even in the spms plant, representative genes encoding heat shock proteins (HSPs) and heat shock transcription factors were upregulated upon HS, while the expression of such genes was increased in a positively correlated manner with Spm content. Furthermore four kinds of HSPs (HSP101, HSP90, HSP70 and HSP17.6) were detected proportionally with the levels of their respective transcripts upon HS. We propose that Spm increases the HS response at transcriptional and translational levels and protects host plants from HS-induced damage.