Eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) are important for leukocyte function. This study investigated whether consuming transgenic Camelina sativa (tCSO) seed oil containing both 20:5n-3 and 22:6n-3 is as effective as fish oil (FO) for increasing the 20:5n-3 and 22:6n-3 content of leukocytes and altering mitogen-induced changes to the T cell transcriptome. Healthy adults (n = 31) consumed 450 mg/day of 20:5n-3 plus 22:6n-3 from either FO or tCSO for 8 weeks. Blood was collected before and after the intervention. 20:5n-3 and 22:6n-3 incorporation from tCSO into immune cell total lipids was comparable to FO. The relative expression of the transcriptomes of mitogen-stimulated versus unstimulated T lymphocytes in a subgroup of 16 women/test oil showed 4390 transcripts were differentially expressed at Baseline (59% up-regulated), 4769 (57% up-regulated) after FO and 3443 (38% up-regulated) after tCSO supplementation. The 20 most altered transcripts after supplementation differed between test oils. The most altered pathways were associated with cell proliferation and immune function. In conclusion, 20:5n-3 and 22:6n-3 incorporation into immune cells from tCSO was comparable to FO and can modify mitogen-induced changes in the T cell transcriptome, contingent on the lipid matrix of the oil.