Docosahexaenoic acid (DHA) is an essential fatty acid modulating key nervous system functions, including neuroinflammation, and regulation of pre- and postsynaptic membrane formation. DHA concentration decreases in the lumbar spinal cord (LSC) of amyotrophic lateral sclerosis (ALS) patients and murine preclinical models. Using a dietary supplementation, we increased DHA levels (2% mean increase, p < 0.01) in the LSC of the familial ALS murine model B6SJL-Tg(SOD1*G93A)1Gur/J. This DHA-enriched diet significantly increases male mouse survival by 7% (average 10 days over 130 days of life expectancy), and delays motor dysfunction (based on stride length) and transgene-associated weight loss (p < 0.01). DHA supplementation led to an increased anti-inflammatory fatty acid profile (ca 30%, p < 0.01) and a lower concentration of circulating proinflammatory cytokine TNF-α (p < 0.001 in males). Furthermore, although DHA-treated mice did not exhibit generally decreased protein oxidative markers (glutamic and aminoadipic semialdehydes, carboxyethyllysine, carboxymethyllysine, and malondialdehydelysine), dietary intake of DHA reduced immunoreactivity towards DNA oxidative damage markers (8-oxo-dG) in the LSC. In vitro we demonstrate that DHA and α-tocopherol addition to a model of motor neuron demise (neonatal rat organotypic spinal cord model under chronic excitotoxicity) also preserves motor neuron number, in comparison with untreated spinal cords. Also, beneficial effects on cell viability were evidenced for the motor neuron cell line NSC-34 in front of H2O2 insult (p < 0.001). Globally we show a sex-specific benefit of dietary DHA supplementation in the G93A ALS mouse model, compared with mice fed an isocaloric control or a n-3-depleted diet. These changes were associated with an increased DHA concentration in the LSC and were compatible with in vitro results showing DHA neuroprotective properties. These results suggest the need for further study on the interaction of gender-influenced biological parameters and DHA in ALS pathogenesis.