To improve our understanding of the disturbed metabolic pathways and cellular responses triggered by honeybee venom stimulation, we compared the changes in serum metabolites in rats, either stimulated or not by honeybee venom, by performing H nuclear magnetic resonance (NMR) spectrometry-based metabonomics to identify potential biomarkers. In this study, 65 metabolites were structurally confirmed and quantified and the following results were obtained. First, by pattern recognition analysis, 14 metabolites were selected as potential biomarkers 3 h after venom stimulation. Second, metabolic pathway analysis showed that methane metabolism, glyoxylate and dicarboxylate metabolism, tricarboxylic acid cycle, glycine, serine, and threonine metabolism, arginine and proline metabolism were affected. Finally, the time-dependent metabolic modifications indicated that rats could recover without medical treatment 24 h after venom stimulation. In summary, this new insight into the changes in serum metabolites in rats after honeybee venom stimulation has enhanced our understanding of the response of an organism to honeybee venom.