This study aimed to investigate whether n-3 polyunsaturated fatty acids (n-3 PUFA) supplementation enhances the anabolic signaling response, muscle hypertrophy, and strength gains following a resistance training program in healthy young men with habitual protein intake ≥1.6g·kg⁻¹·day⁻¹. In a randomized, double-blind, placebo-controlled trial, 46 resistance-trained men (28.1±5.8years, 24.8±3.1kg/m2, 1.8±0.5g·kg-1·day-1 protein intake) were assigned to receive either n-3 PUFA (W3; 6.3g/day n-3PUFA) or placebo (PLA) during 14 weeks of supervised lower-limb resistance training 2x/week. Body composition, ultrasound-derived muscle cross-sectional area (mCSA), maximal dynamic strength (1-RM), muscle tissue fatty acid composition, and muscle fiber morphology were assessed pre- and post-intervention. Phosphorylation of mTOR, p70S6K, and 4E-BP1 were evaluated at rest and 4 h after exercise, before and after the intervention. Adherence to supplementation and training was high in both groups (>94% n-3 PUFA supplementation; >96% training session attendance). A marked incorporation of n-3 PUFA into skeletal muscle (interaction p=0.017) was observed in the W3 group. Both groups presented increases in lean mass, 1-RM strength, rectus femoris and vastus lateralis mCSA, vastus lateralis muscle fiber CSA, and minimum Feret’s diameter (all main time effects p<0.05), with no differences between groups. No effects were observed for anabolic signaling between groups or over time (all p>0.05). In conclusion, despite substantial incorporation into muscle tissue, n-3 PUFA supplementation did not enhance muscle hypertrophy, strength gains, or anabolic signaling in healthy adult men with adequate protein intake undergoing resistance training. Future studies should focus on populations with impaired anabolic responses, during periods of disuse or caloric restriction.