Methylglyoxal (MG) and calcium ion (Ca2+) can increase multiple-stress tolerance including plant thermotolerance. However, whether crosstalk of MG and Ca2+ exists in the formation of maize thermotolerance and underlying mechanism still remain elusive. In this paper, maize seedlings were irrigated with MG and calcium chloride alone or in combination, and then exposed to heat stress (HS). The results manifested that, compared with the survival percentage (SP, 45.3%) of the control seedlings, the SP of MG and Ca2+ alone or in combination was increased to 72.4%, 74.2%, and 83.4% under HS conditions, indicating that Ca2+ and MG alone or in combination could upraise seedling thermotolerance. Also, the MG-upraised SP was separately weakened to 42.2%, 40.3%, 52.1%, and 39.4% by Ca2+ chelator (ethylene glycol tetraacetic acid, EGTA), plasma membrane Ca2+ channel blocker (lanthanum chloride, LaCl3), intracellular Ca2+ channel blocker (neomycin, NEC), and calmodulin (CaM) antagonist (trifluoperazine, TFP). However, significant effect of MG scavengers N-acetylcysteine (NAC) and aminoguanidine (AG) on Ca2+-induced thermotolerance was not observed. Similarly, an endogenous Ca2+ level in seedlings was increased by exogenous MG under non-HS and HS conditions, while exogenous Ca2+ had no significant effect on endogenous MG. These data implied that Ca2+ signaling, at least partly, mediated MG-upraised thermotolerance in maize seedlings. Moreover, the activity and gene expression of glyoxalase system (glyoxalase I, glyoxalase II, and glyoxalase III) and non-glyoxalase system (MG reductase, aldehyde reductase, aldo-keto reductase, and lactate dehydrogenase) were up-regulated to a certain extent by Ca2+ and MG alone in seedlings under non-HS and HS conditions. The up-regulated MG-scavenging system by MG was enhanced by Ca2+, while impaired by EGTA, LaCl3, NEC, or TFP. These data suggest that the crosstalk of MG and Ca2+ signaling in maize thermotolerance through MG-scavenging system. These findings provided a theoretical basis for breeding climate-resilient maize crop and developing smart agriculture.