High-latitude marine environments, such as the Beagle Channel, are highly vulnerable to modifications related to climate change and anthropogenic impacts, which can rapidly affect the ecological attributes of plankton communities and thus alter ecosystem trophodynamics. This study compares the mesozooplankton community structure in the inner and outer (eastern) Beagle Channel during spring and assesses the interactions of the main mesozooplankton trophic groups with lower and higher trophic level organisms under an isotopic approach. Oceanographic data and biological samples, ranging from sediment and phytoplankton to pelagic squat lobsters (Grimothea gregaria), were collected along the channel during two research cruises conducted in November 2019. Mesozooplankton abundance and taxonomic composition differed between sectors, in agreement with their distinct environmental conditions. The inner sector was dominated by copepods, mainly Clausocalanidae spp., followed by cirripede and echinoderm larvae and appendicularians. In the outer sector, with shallower, saltier, and warmer waters than the inner one, mesozooplankton abundances were notably higher and both copepods and decapod larvae, mainly Peltarion spinosulum and G. gregaria zoeae, were dominant. This pattern in meroplankton abundance was consistent with the bathymetric distribution reported for benthic adults. Between-sector differences in taxonomic composition, e.g., Macruronus novaezelandiae (hake) larvae occurring only in the outer sector, may be attributed to the higher connectivity between this sector and the open ocean and the semi-enclosed character of the inner channel. Mesozooplankton spatial variability was partially reflected in the isotopic niche width of their different trophic groups such as copepods, euphausiids and decapod larvae. However, at the community level, trophic attributes (i.e., baseline resources, trophic positions, isotopic diversity metrics) were quite similar in the two sectors, suggesting similar basal and vertical trophic structures. According to Bayesian stable isotope mixing models, most trophic groups, including pelagic G. gregaria, rely on phytoplankton as their main carbon source. This reveals a weak predation pressure on mesozooplankton by the squat lobster and reinforces a bottom-up regulation during the spring season. This study contributes to our knowledge of trophic interactions in plankton communities and how they are regulated by bottom-up and top-down forces, which is imperative for monitoring and management purposes.