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BACKGROUND: Macrocyclic lactones are arguably the most successful chemical class with efficacy against parasitic nematodes. Here we investigated the effect of the macrocyclic lactone ivermectin on lipid homeostasis in the plant parasitic nematode Globodera pallida and provide new insight into its mode of action. RESULTS: A non-invasive, non-destructive, label-free and chemically selective technique called Coherent anti-Stokes Raman scattering (CARS) spectroscopy was used to study lipid stores in G. pallida. We optimised the protocol using the free-living nematode Caenorhabditis elegans and then used CARS to quantify lipid stores in the pre-parasitic, non-feeding J2 stage of G. pallida. This revealed a concentration of lipid stores in the posterior region of J2s within 24 hours of hatching which decreased to undetectable levels over the course of 28 days. We tested the effect of ivermectin on J2 viability and lipid stores. Within 24 hours ivermectin paralysed J2s. Counter-intuitively, over the same time-course ivermectin increased the rate of depletion of J2 lipid, suggesting in ivermectin-treated J2s there is a disconnection between the energy requirements for motility and metabolic rate. This decrease in lipid stores would be predicted to negatively impact on J2 infective potential. CONCLUSION: These data suggest that the benefit of macrocyclic lactones as seed treatments may be underpinned by a multilevel effect involving both neuromuscular inhibition and acceleration of lipid metabolism.