ABSTRACTOrganophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We hypothesized that dietary supplementation withLactobacillus, a genus of commensal bacteria, would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and chlorpyrifos [CP]). SeveralLactobacillusspecies were screened for toleration of 100 ppm of CP or parathion in MRS broth based on 24-h growth curves. CertainLactobacillusstrains were unable to reach stationary-phase culture maxima and displayed an abnormal culture morphology in response to pesticide. Further characterization of commonly used, pesticide-tolerant and pesticide-susceptible, probioticLactobacillus rhamnosusstrain GG (LGG) andL. rhamnosusstrain GR-1 (LGR-1), respectively, revealed that both strains could significantly sequester organophosphate pesticides from solution after 24-h coincubations. This effect was independent of metabolic activity, asL. rhamnosusGG did not hydrolyze CP and no difference in organophosphate sequestration was observed between live and heat-killed strains. Furthermore, LGR-1 and LGG reduced the absorption of 100 μM parathion or CP in a Caco-2 Transwell model of the small intestine epithelium. To determine the effect of sequestration on acute toxicity, newly eclosedDrosophila melanogasterflies were exposed to food containing 10 μM CP with or without supplementation with live LGG. Supplementation with LGG simultaneously, but not with administration of CP 3 days prior (prophylactically), mitigated CP-induced mortality. In summary, the results suggest thatL. rhamnosusmay be useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings could be transferable to clinical and livestock applications due to affordability and practical ability to supplement products with food-grade bacteria.IMPORTANCEThe consequences of environmental pesticide pollution due to widespread usage in agriculture and soil leaching are becoming a major societal concern. Although the long-term effects of low-dose pesticide exposure for humans and wildlife remain largely unknown, logic suggests that these chemicals are not aligned with ecosystem health. This observation is most strongly supported by the agricultural losses associated with honeybee population declines, known as colony collapse disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial microorganisms in fermented foods and have shown potentials to sequester and degrade environmental toxins. This study demonstrated that commonly used probiotic strains of lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and chlorpyrifos). ThisLactobacillus-mediated sequestration was associated with decreased intestinal absorption and insect toxicity in appropriate models. These findings hold promise for supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce organophosphate pesticide exposure.