Due to the side effects of chemical drugs in pain relief, the use of herbal medicines is increasing. Red algae have antioxidant, antibacterial, antifungal, antitumor, and antimicrobial properties. The present study aimed to investigate the effect of Red Algae ethanolic extract and its possible neural interactions on formalin-induced pain in mice. 125 adult male mice allocated in 6 experiments. In the first experiment, mice were injected intraperitoneally with saline, red algae extract (2. 5, 5, and 10 mg/kg), and morphine (5 mg/kg). In the second experiment, mice received intraperitoneal injections of saline, red algae extract (10 mg/kg), naloxone (non-selective opioid antagonist, 2 mg/kg), and naloxone + red algae extract. Experiments 4 to 6 were similar to the second experiment but injected with flumazenil (non-selective GABA receptor antagonist, 5 mg/kg), cyproheptadine (non-selective serotonin receptor antagonist, 2 mg/kg), chlorpheniramine (histamine H1receptors antagonist, 20 mg/kg) and cimetidine (histamine H2receptors antagonist, 12. 5 mg/kg) were used instead of naloxone. After 30 minutes, subcutaneous injection of formaldehyde was performed on the plantar surface of the right foot, and the time of licking, chewing, and biting (Licking Time) was measured. According to the results, morphine significantly reduced pain time compared to the control group (P<0. 05). Red algae extract in a dose-dependent manner reduced pain compared to the control group (P<0. 05). Injection of naloxone + red algae extract significantly reduced the analgesic effects of red algae extract compared to the group of red algae extract (P<0. 05). Flumazenil + red algae extract significantly reduced the analgesic effects of red algae extract (P<0. 05). Injection of cyproheptadine + red algae extract significantly enhanced the analgesic effects of red algae extract (P<0. 05). Injection of chlorpheniramine + red algae extract significantly reduced the analgesic effects of red algae extract (P<0. 05). The results showed that the analgesic effects of red algae are mediated by opioidergic, serotonergic, GABAergic and histaminergic pathways.