The gas phase extract of mainstream smoke of combustion cigarettes includes many carbonyl compounds, which increase oxidative stress. The present study examined whether carbonyl compounds in heated cigarette-derived smoke extract (hCSE) and burned cigarette-derived smoke extract (bCSE) cause a reduction in endothelial nitric oxide synthase (eNOS) activity. Three types of heat-not-burn cigarettes (Ploom S, glo, and IQOS) and a combustion cigarette (hi-lite) were used to generate cigarette smoke at different heating or combustion temperatures [Ploom S (200°C), glo (240°C), IQOS (300–350°C), and hi-lite (770−870℃)]. The amounts of carbonyl compounds in hCSE/bCSE were assessed by measuring the carbonylation level of Ca²⁺-sensing receptor (CaSR) that promotes the phosphorylation of eNOS at Ser¹¹⁷⁷, which positively regulates eNOS activity. Although CaSR-mediated phosphorylation of eNOS were unaffected by hCSE from Ploom S and glo, hCSE/bCSE from IQOS and hi-lite reduced the eNOS phosphorylation. hCSE/bCSE from the cigarettes, except for that from Ploom S, facilitated carbonylation of CaSR with different potencies (rank order: glo < IQOS < hi-lite). The reduction of eNOS phosphorylation and the carbonylation of CaSR induced by hCSE/bCSE from IQOS and hi-lite were inhibited by treatment with mainstream smoke using a Carboxen-572 cartridge to scavenge carbonyl compounds. These results suggest that an increase in the heating or combustion temperature leads to an increase in the generation of carbonyl compounds, which cause endothelial dysfunction characterized by a reduction in eNOS activity.