Neonicotinoids (NEOs) have become the most widely used insecticides in the world since the mid-1990s. According to Chinese dietary habits, rice and water are usually heated before being consumed, but the information about the alteration through the heat treatment process is very limited. In this study, NEOs in rice samples were extracted by acetonitrile (ACN) and in tap water, samples were extracted through an HLB cartridge, then, a high-performance liquid chromatography system and a triple quadrupole mass spectrometry (HPLC-MS/MS) were applied for target chemical analysis. The parents of NEOs (p-NEOs) accounted for >99% of the total NEOs mass (∑NEOs) in both uncooked (median: 66.8 ng/g) and cooked (median: 41.4 ng/g) rice samples from Guangdong Province, China, while the metabolites of NEOs (m-NEOs) involved in this study accounted for less than 1%. We aimed to reveal the concentration changes of NEOs through heat treatment process, thus, several groups of rice and water samples from Guangdong were cooked and boiled, respectively. Significant (p< 0.05) reductions in acetamiprid, imidacloprid (IMI), thiacloprid, and thiamethoxam (THM) have been observed after the heat treatment of the rice samples. In water samples, the concentrations of THM and dinotefuran decreased significantly (p< 0.05) after the heat treatment. These results indicate the degradation of p-NEOs and m-NEOs during the heat treatment process. However, the concentrations of IMI increased significantly in tap water samples (p< 0.05) after heat treatment process, which might be caused by the potential IMI precursors in those industrial pesticide products. The concentrations of NEOs in rice and water can be shifted by the heat treatment process, so this process should be considered in relevant human exposure studies.