Colchicine (CHC), a poisonous plant alkaloid, has been widely utilized for decades in the treatment of gout, but has a rather low therapeutic index, which causes oxidative stress leading to cognitive impairment, brain damage, apoptosis, and hitopathological alterations in humans and experimental animals. The present investigation evaluated the potential palliative effect of the pumpkin seeds oil (PSO) at a dose of 4 ml/kg b.wt against CHC (0.6 mg/kg b.wt) -induced neurotoxic and neurobehavioral effects in rats. Forty male rats weighing 245–260 g were assigned to four groups. The results displayed that CHC exposure induced neurobehavioral disorders and a remarkable decline in the serotonin and dopamine levels and the immunoexpression of BDNF and GFAP in the brain. Besides, CHC treatment evoked brain oxidative stress, as manifested by depleted antioxidant enzyme activities and elevated malondialdehyde (MDA) and protein carbonyl (PC) levels. Also, CHC triggered brain DNA damage, as indicated by a marked increment in the brain 8-Hydroxyguanosine (8-OHdG) level. However, concurrent treatment with the PSO effectively attenuated the CHC-induced toxic effects as evidenced by a noticeable increase in the serotonin (33 ± 3.05) and dopamine (2.48 ± 0.40) concentrations, and the BDNF and GFAP immunoexpression in the brain. Moreover, PSO mitigated CHC-induced brain oxidative stress and DNA damage as shown by elevated antioxidant enzyme activities (164 ± 3.46 SOD and 7.55 ± 0.43 CAT) and reduced MDA (1.62 ± 0.23), PC (1.35 ± 0.23), and 8-OHdG (3.02 ± 0.33) levels. These results concluded that PSO could serve as a therapeutic strategy to ameliorate the neurotoxic and neurobehavioral impacts of CHC.