Fatty acid (FA) profile and stable isotope (SI) signature analyses has been used to examine feeding ecology of Antarctic toothfish and food webs of Antarctic marine ecosystem. Specimens of Antarctic toothfish, by-catch and their stomach contents were collected for the past five years in the eight locations of he Antarctic Waters. We investigated ontogenetic and spatial variability in resource utilization of Antarctic toothfish and distribution patterns of FA profiles in various organisms because of the particular importance of accumulation of lipid deposits in their tissue. Toothfish trophic niche indices was calculated using both FA profiles and SI values. Results shows spatial variation in their resource utilization between the Ross Sea shelf (RSS) and the deep-sea slopes the western Indian Ocean sector (IOS) and the slope towards the Pacific Ocean sector (POS). FA profiles of toothfish were characterized by high proportions of monounsaturated FAs and low proportions of polyunsaturated FAs, differing from other organisms in their habitats. Distribution patterns of FA profiles among various organisms were well established by self-organizing map (SOM) analysis. Similar FA profiles and SI values of toothfish between ISO and POS indicate a depth-dependent dietary difference from that of RSS. This appears to be closely associated with ontogenetic and spatial variation in resource utilization during their migration. The combination of both tracers explained well their dietary utilization pattern. Indeed, a recently developed mixing model calculation for FA profiles and SI values, which follows the Bayesian machiner, highlights the nutritional importance of Noththenidae fish compared to Channichthyidae species to whole population level. In contrast, the model calculations provided an estimation of dietary shift during ontogeny and thereby migration to deep water. Finally, the model results clearly explained spatial shifts on the nonmetric multidimensional scaling plot and the SOM map by realistic diet proportions.