Abstract Objectives This study was conducted to evaluate the effects of exposure to pioglitazone, a peroxisome proliferator-activated receptor agonist, on cognitive impairment induced by scopolamine, a muscarinic antagonist, in mice. Methods Pioglitazone (9 mg/kg, 18 mg/kg) was orally administered for 9 days at 30 min before intraperitoneal injection with scopolamine (0.8 mg/kg, i.p.). Cognitive function was evaluated by the passive avoidance test and the Morris water maze test on the 10th day after treatment. Changes in cholinergic system reactivity were also examined by measuring the acetylcholine, acetylcholinesterase and choline acetyltransferase in the hippocampus and cortex. Key findings Scopolamine injection induced impaired performance in the passive avoidance test and the water maze test and severe decrease of cholinergic system reactivity, as indicated by reduced acetylcholine levels, decreased choline acetyltransferase activity and increased acetylcholinesterase activity. Daily administration of pioglitazone significantly increased step-through latency in passive avoidance test, and significantly decreased the escape latency, and increased the time spent in the platform quadrant in the Morris water maze test. Pioglitazone also protected against scopolamine-induced cholinergic system deficit, including reduced acetylcholine levels, decreased choline acetyltransferase activity and increased acetylcholinesterase activity in the hippocampus or cortex. Conclusions Pioglitazone demonstrates a significant neuroprotective effect against scopolamine-induced cholinergic system deficit and cognitive impairment.