Objective To compare the serum small molecule metabolites between heat acclimatization and heat non-acclimatization subjects, and reveal the characteristics of serum small molecule metabolites related to human heat acclimatization. Methods Seventy subjects involved in a cold-to-heat region transferring were enrolled in this study. According to the exposure to a high wet-heat natural environment or a comfortable air-condition artificial environment, the subjects were divided into heat acclimatization group (n＝40) and heat non-acclimatization group (n＝30). Early morning fasting blood samples from the subjects were collected 2 months after region transferring. After standard processing, liquid chromatography tandem mass spectrometry was used for metabolomic detection, and Progenesis QI software and ropls tool of R 1.6.2 software were employed to identify the differential small molecule metabolites of the 2 groups, and the pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was carried out for the differential small molecule metabolites. Results There were no significant differences in age or body mass index between the 2 groups (both P＞0.05). A total of 1 320 small molecule metabolites were detected by serum metabolomics. Orthogonal partial least squares-discriminant analysis showed that the samples of heat acclimatization group and heat non-acclimatization group had a good discrimination between groups and a good aggregation within groups. There were 89 small molecule metabolites with a concentration difference of more than 1.5 times (P＜0.05) between the 2 groups, with 57 being increased and 32 being decreased in the heat acclimatization group. KEGG pathway enrichment analysis showed that the differential small molecule metabolites were mainly enriched in 13 metabolic pathways, including amino acid metabolism and glycerophospholipid metabolism, involving tryptophan, glycine, serine, threonine, arginine, and proline. Conclusion Heat acclimatization training under heat exposure can change the omics characteristics of small molecule metabolites in human serum, mainly enhancing amino acid metabolism involved in heat tolerance.