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A low intake of long-chain polyunsaturated ω3 fatty acid often prevails in Western populations. Its consequences in terms of the control of fuel homeostasis led us to explore functional events in pancreatic islets isolated from either normal or ω3-depleted rats (second generation). In the latter rats, the inflow of K+by both ouabain-sensitive and ouabain-resistant modalities was decreased, this coinciding with an impaired insulin secretory response to ouabain. The intravenous injection of a medium-chain triglyceride: fish oil emulsion to ω3-depleted rats 2 h before sacrifice restored a normal value for the inflow of K+by the ouabain-sensitive modality, i.e., that linked to the activity of the Na,K-ATPase, but failed to correct the entry of K+by the ouabain-resistant modality and the defect of the insulin secretory response to ouabain. In conclusion, an impaired activity of the Na,K-ATPase in insulin-producing cells apparently represents a key determinant of altered islet function in ω3-depleted rats.