Objectives: The flower from chrysanthemum (Chrysanthemum indicum L.; CI) has been used in China as traditional Chinese medicine (TCM) for various symptoms; for example, hypertension, vertigo and infections. It has high potencies in several pharmacological effects including anti-inflammatory, antipyretic and analgesic activities. Serotonin (5-HT) is found in both central and peripheral nervous system. It plays a role in the process of pain, especially, severe pain such as migraine. Therefore, the purpose of this study aimed to evaluate the effect of CI on pain relieving and brain 5-HT in rat model. Methods: The ethanolic extract of white chrysanthemum flower was used in this study. Rats were pretreated with CI at 250, 500, and 750 mg/kg, 200 mg/kg acetaminophen, and 100 mg/kg ibuprofen compared with vehicle (control). Tail-flick meter was used to determine analgesic effect. Comparisons of tail-flick latency and % maximal possible effect (MPE) among groups were evaluated. The dose of CI that produced the highest activities in both tail-flick latency and %MPE was then selected to study in reserpine-induced low 5-HT experiment compared with sumatriptan, a standard drug for the migraine treatment, to examine the brain 5-HT. Results: The results showed that 500 mg/kg CI significantly possessed analgesic activity in rats as well as acetaminophen and ibuprofen, standard painkillers, did. Therefore, CI at this dose was chosen to study in reserpine-rat experiment. The study demonstrated that CI500 statistically boosted the brain 5-HT levels in reserpine-induced low 5-HT rats. An increase in the brain 5-HT was also found in rats treated with sumatriptan, a 5-HT agonist. It was possible that CI acted as “5-HT agonist” similarly to sumatriptan. Conclusion: These findings suggest that the effect of CI on the minimization of algesia is possibly involved in serotonergic system. As it affected both pain behavior and brain 5-HT level, CI could be useful to be developed as an alternative antihyperalgesic substance, particularly migraine. © 2016, Faculty of Pharmaceutical Sciences, Chulalongkorn University. All rights reserved.