The aim of this study was to evaluate whether thymosin β4 (Tβ4) levels are increased in the serum of rheumatoid arthritis (RA) patients, and if this increase is associated with RA disease activity and resistance to treatment. Blood samples from 40 patients with RA were collected at baseline and 6 months after starting treatment with disease-modifying antirheumatic drugs (DMARD) and/or tumor necrosis factor (TNF)-α blocker. Serum levels of Tβ4 were measured by ELISA. Tβ4 levels (mean ± standard deviation) in RA patients were significantly (approximately tenfold) higher than in healthy controls (577.4 ± 67.92 vs. 56.61 ± 5.72 ng/mL). Serum Tβ4 levels in patients with severe disease activity before therapy were slightly higher than in patients with moderate disease activity (662.4 ± 491.5 vs. 462.5 ± 305.3 ng/ml, P > 0.05). Tβ4 levels were significantly associated with disease activity according to the 28-joint Disease Activity Score. The mean Tβ4 level at baseline in the DMARD treatment group was significantly lower than in the DMARD + TNF-α blocker treatment group. Tβ4 levels were increased in the serum of patients with RA and were positively associated with disease activity. Levels of Tβ4 may also be relevant in determining or predicting resistance to RA treatment. Further studies are necessary to determine if Tβ4 is an appropriate therapeutic target for controlling inflammation associated with RA.