Abstract Midwater zooplankton are major agents of biogeochemical transformation in the open ocean; however their characteristics and activity remain poorly known. Here we evaluate midwater zooplankton biomass, amino acid (AA)-specific stable isotope composition (δ15N values) using compound-specific isotope analysis of amino acids (CSIA-AA), trophic position, and elemental composition in the North Pacific Subtropical Gyre (NPSG). We focus on zooplankton collected in the winter, spring, and summer to evaluate midwater trophic dynamics over a seasonal cycle. For the first time we find that midwater zooplankton respond strongly to seasonal changes in production and export in the NPSG. In summer, when export from the euphotic zone is elevated and this ‘summer pulse’ material is transported rapidly to depth, CSIA-AA indicates that large particles (>53 μm) dominate the food web base for zooplankton throughout the midwaters, and to a large extent even into the upper bathypelagic zone. In winter, when export is low, zooplankton in the mid-mesopelagic zone continue to rely on large particle basal resources, but resident zooplankton in the lower mesopelagic and upper bathypelagic zones switch to include smaller particles (0.7–53 μm) in their food web base, or even a subset of the small particle pool. Midwater zooplankton migration patterns also vary with season, with migrants distributed more evenly at night through the euphotic zone in summer as compared to being more compressed in the upper mixed layer in winter. Deeper zooplankton migration within the mesopelagic zone is also reduced in late summer, likely due to the increased magnitude of large particle material available at depth during this season. Our observed seasonal change in activity and trophic dynamics drives modestly greater biomass in summer than winter through the mesopelagic zone. In contrast midwater zooplankton carbon (C), nitrogen (N), and phosphorus (P) composition does not change with season. Instead we find increasing C:N, C:P, and N:P ratios with greater depths, likely due to decreases in proteinaceous structures and organic P compounds and increases in storage lipids with depth. Our study highlights the importance and diversity of feeding strategies for small zooplankton in NPSG midwaters. Many small zooplankton, such as oncaeid and oithonid copepods, are able to access small particle resources at depth and may be an important trophic link between the microbial loop and deep dwelling micronekton species that also rely on small particle-based food webs. Our observed midwater zooplankton trophic response to export-driven variation in the particle field at depth has important implications for midwater metabolism and the export of C to the deep sea.