Pain, an intricate experience encompassing physiological and psychological aspects, often arises from injury, inflammation, and emotional distress. Current pain management, primarily reliant on opioids, has limitations. Non-opioid medications like non-steroidal anti-inflammatory drugs (NSAIDs) offer relief but pose gastrointestinal and cardiovascular risks. This study aimed to elucidate the possible antinociceptive mechanism of the ethanolic extract of Borago officinalis flower (EEBO) examined in vivo using ?2-adrenergic (Yohimbine), adenosinergic (Caffeine), dopaminergic (Haloperidol), serotonergic (Ondansetron) , cholinergic (atropine) receptors , GABAergic (Flumanzil) , opioid (Naloxone), potassium channels (Glibenclamide) and calcium channels (Nifidipine) in acetic acid induced writhing in mice. Moreover, EEBO flower tested against formalin , glutamate , capsaicin, and phorbol 12-myristate 13-acetate-induced nociception models. Additionally , the locomotor activity of EEBO flower examined using the actophotometer test. Furthermore, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) was employed to analyze the constituents of EEBO flowers, revealing that ?-linolenic acid, stearic acid, and linoleic acid were the major compounds, respectively. This study found that the antinociceptive activity of EEBO flower (400 mg/kg i.p) administration was significantly inhibited by Ondansetron, Yohimbine, Flumazenil, Glibenclamide , Also , antinociceptive activity of EEBO flower have significant inhibition in formalin induced nociceptive model. Moreover , Locomotor test exhibited no significant impact on sensorimotor activity. In conclusion , EBBO flower has affinity on serotonergic, adrenergic, and GABAergic receptors and ATP-sensitive K+ channels. Also , EEBO flower showed no significant difference on early phase, but showed significant difference on late phase in formalin-mediated nociceptive pathways. As a result, the presence of many bioactive chemicals of the EEBO flower may be associated with the antinociceptive activity. Keywords : Borago officinalis L. , antinociceptive , serotonergic, adrenergic, and GABAergic receptors and ATP-sensitive K+ channels