To investigate whether conjugated salicylic acid (SA)—SA 2-O-β-D-glucose (SAG) involves thermotolerance of pea leaves induced by heat acclimation, we used the inhibitor of SA biosynthesis (i.e., paclobutrazol; PAC) to evaluate the role of SAG, and found that PAC treatment reduced the thermotolerance of pea leaves induced by heat acclimation. Our further analyses indicated that PAC pretreatment led to an abrupt decrease in SAG during heat acclimation, which might be not only due to the inhibition of SA biosynthesis but also to the conversion retarding of free SA to SAG catalyzed by glucosyltransferase (SAGT). These results suggest that SAG may act as a signal molecule as SA does, to involve thermotolerance induced by heat acclimation.