Background The non-catalytic region of tyrosine kinase (Nck) is an adaptor protein, which is ubiquitously expressed in many types of cells. In T cells, the Nck1 isoform promotes T cell receptor signalling as well as actin polymerisation. However, the role of Nck1 in the lipid metabolism in T cells is unknown. In the present study, we investigated the effect of the Nck1 protein and Nck–CD3 interaction on lipid metabolism and on the physical and biological properties of Jurkat T cells, using a newly developed holotomographic microscope. Results Holotomographic microscopy showed that Nck1-knocked-out cells had membrane blebs and were irregular in shape compared to the rounded control cells. The cell size and volume of Nck1-deficient cells were comparable to those of the control cells. Nck1-knocked-out Jurkat T cells had a greater lipid content, lipid mass/cell mass ratio, and lipid metabolite levels than the control cells. Interestingly, treatment with a small molecule, AX-024, which inhibited Nck–CD3 interaction, also caused an increase in the lipid content in wild-type Jurkat T cells, as found in Nck1-deficient cells. Conclusions Knockout of Nck1 protein and hindrance of the Nck–CD3 interaction cause the elevation of lipid content in Jurkat T cells.