High doses of malathion, an organophosphorus (OP) insecticide ubiquitously used in agriculture, residential settings, and public health programs worldwide, induce a well-defined toxidrome that results from the inhibition of acetylcholinesterase (AChE). However, prenatal exposures to malathion levels that are below the threshold for AChE inhibition have been associated with increased risks of neurodevelopmental disorders, including autism spectrum disorder with intellectual disability comorbidity. The present study tested the hypothesis that prenatal exposures to a non-AChE-inhibiting dose of malathion are causally related to male-biased cognitive deficits later in life in a precocial species. To this end, pregnant guinea pigs were injected subcutaneously with malathion (20 mg/kg) or vehicle (peanut oil, 0.5 ml/kg) once daily between approximate gestational days 53 and 63. This malathion dose regimen caused no significant AChE inhibition in the brain or blood of dams and offspring and had no significant effect on the postnatal growth of the offspring. Around postnatal day 30, locomotor activity and habituation, a form of non-associative learning, were comparable between malathion- and peanut oil-exposed offspring. However, in the Morris water maze, malathion-exposed offspring presented significant sex-dependent spatial learning deficits in addition to memory impairments. These results are far reaching as they indicate that: (i) malathion is a developmental neurotoxicant and (ii) AChE inhibition is not an adequate biomarker to derive safety limits of malathion exposures during gestation. Continued studies are necessary to identify the time and dose dependence of the developmental neurotoxicity of malathion and the mechanisms underlying the detrimental effects of this insecticide in the developing brain.Copyright © 2020. Published by Elsevier Inc.