Tumor necrosis factor alpha-related apoptosis inducing ligand (TRAIL) is a pro-apoptotic factor that can also induce cell proliferation. The role of TRAIL in rheumatoid arthritis (RA) is still unclear. Previously, we reported that TRAIL induces RA fibroblast-like synoviocyte (FLS) proliferation. We now investigate the intracellular mechanisms involved in TRAIL-induced cell proliferation. Therefore, we tested the effect of TRAIL on signaling pathways including MAP kinases (p38 and ERK1/2) and PI3 kinase/Akt, known to control cell proliferation. For all these experiments, the concentration of TRAIL used to stimulate cell was 0.5 nM. TRAIL induced p38 MAP kinase but with a lower intensity in comparison with tumor necrosis factor alpha (TNF-α) (n = 3). TRAIL also induced ERK1/2 and Akt phosphorylation in RA FLS in a time-dependent manner, showing maximum activation between 5 and 10 min by western blot (n = 3). This kinetic and intensity of ERK and Akt activation were similar to positive control TNF-α (n = 3). The transcription factor NF-κB plays a major role in cell survival and is activated by MAP kinases and Akt. When NF-κB is activated, it translocates from the cytoplasm to the nucleus. We examined NF-κB translocation into the nucleus of RA FLS following TRAIL stimulation using immunofluorescence. RA FLS treated with TRAIL did not induce a nuclear uptake of NF-κB (n = 3). The percentages of RA FLS-positive NF-κB translocation was increased from 4.3 ± 0.9% to 8.9 ± 2.3% when stimulated with TRAIL. For the positive control IL-1β, this percentage increased to 97.9 ± 1.1%. This result was confirmed by western blot. To determine the implication of MAP kinases, and PI3 kinase/Akt in TRAIL-induced proliferation, we therefore tested different specific signaling inhibitors including PI3 kinase inhibitor LY294002, ERK1/2 inhibitor PD98059, and p38 inhibitor SB203580. RA FLS were pretreated with the specific inhibitors at different concentrations or Me2SO vehicle control for 1 hour and were then stimulated with TRAIL. Proliferation was assessed according to the level of incorporated tritiated thymidine. ERK 1/2 inhibitor PD98059 and p38 inhibitor SB203580 significantly downregulated TRAIL-induced proliferation in a dose-dependent manner (n = 3). PI3 kinase inhibitor LY294002 almost completely blocked proliferation promoted by TRAIL. These results highlight the main role of Akt in TRAIL-mediated proliferation.