Mixotrophic nanoflagellates (MNFs) are key players in Arctic marine food webs, but their contribution to bacterivory across depth gradients remains poorly quantified. Here, we conducted depth-resolved grazing experiments in the Pacific Arctic Ocean to quantify the vertical distribution and bacterivorous activity of MNFs and heterotrophic nanoflagellates (HNFs) and to identify the environmental drivers underlying their interactions. Our results demonstrate a pronounced, inverse depth-dependent shift in the dominance of nanoflagellates bacterivory that challenges traditional paradigms. In the euphotic zone, obligate HNFs outcompeted mixotrophs as the primary bacterial consumers, with the 2–5 µm size fraction accounting for up to 81.15% of total HNF abundance. Unburdened by the metabolic costs of maintaining photosynthetic machinery, highly specialized HNFs maximized their grazing rates, tightly tracking bacterial prey availability. Conversely, below the photic zone, HNF abundance and grazing decreased sharply, while MNF populations remained stable despite declining bacterial abundance. Relying on metabolic flexibility and dark survival strategies, MNFs became the dominant grazers in the aphotic zone, where their grazing contribution reached up to 70% of total nanoflagellate bacterivory. These findings highlight a distinct vertical niche partitioning driven by resource allocation trade-offs, demonstrating that mixotrophy is a highly effective strategy for sustaining functional microbial populations under extreme light and prey limitation. By altering the relative contributions of bacterivores across the water column, this depth-partitioned grazing profoundly influences microbial trophic organization, with critical implications for biological carbon transfer in the rapidly changing Arctic Ocean.