(1) Background: In the healthy ageing, NK cell number is not modified; however, their spontaneous cytotoxicity decreases. We postulated that the age-dependent decline in metabolic activities might be responsible for this effect. (2) Methods: The fatty acid profile of 30 healthy young males (23 ± 4 years old, BMI 22.1 ± 1.3) and 30 older males (63 ± 5 years old, BMI 22.9 ± 2.5) donors were evaluated along with the expression of killing (KR) and inhibitory NK receptors (KIR) at basal level and after cultivation with fatty acids for 24 h. (3) Results: Significantly higher levels of oleic (p < 0.01), arachidonic (p < 0.001), lignoceric (p < 0.001), and nervonic acids (p < 0.0001) and significantly lower levels of docosapentaenoic and docosahexaenoic acids (p < 0.01) were found in elders as compared to young adults. At basal levels, significant (p < 0.005) differences in KR and KIR expression were encountered; 12/16 antigens. Treatment of cells with saturated fatty acids or arachidonic acid (AA) significantly enhanced KR expressions (p < 0.001). AA treatment decreased inhibitory KIR expression while docosahexaenoic, and eicosapentaenoic acid increased them. (4) Conclusions: Changes in fatty acids blood levels, and KR and KIR expression in NK cell, are age-dependent. Supplementation of NK cells with eicosapentaenoic or docosahexaenoic acid enhanced inhibitory KIR receptors’ expression which may improve their cell function.