The region of surface water–groundwater interaction in streams, the hyporheic zone, is important for biogeochemical processes and provides habitat for specialized microbial and invertebrate assemblages. Although hyporheic invertebrates contribute little biomass and respiration relative to microbes in stream sediments, invertebrate effects on biogeochemical processes may be disproportionately large. We tested how various interstitial invertebrate assemblages affected N cycling and respiration in flow-through microcosms filled with alluvial sediment in the laboratory. Average invertebrate biomasses in low and high invertebrate treatments were 0.20 and 19 mg dry mass/L sediment, respectively. Average net NO₃⁻ regeneration/uptake rate increased with increasing invertebrate biomass, showing invertebrates suppressed NO₃⁻ uptake or stimulated in situ NO₃⁻ production. Average respiration (normalized for sediment organic matter) and particulate organic matter (POM) increased 51% and 33%, respectively, with increasing invertebrate biomass, suggesting direct contribution to hyporheic metabolism and/or stimulation of microbial activity and an accumulation of POM driven by invertebrates. We suggest that interstitial invertebrates can substantially alter biogeochemical processes in hyporheic zones.