This study aimed to determine the correlation of disease activity of rheumatoid arthritis (RA) with Th17/regulatory T cell (Treg) and Forkhead box protein 3 (Foxp3) cells ratio in patients under therapy with anti-tumor necrosis factor (TNF)-α. Totally, 84 patients with RA and 13 healthy controls were included in this case-control study. The patients were divided into four groups to receive only methotrexate (MTX) (n=25), monotherapy (anti-TNF) (n=18), and combined therapy (MTX+anti-TNF) (n=26); however, one group received no medications (n=15) and was regarded as a positive control. Other 13 healthy controls that were considered negative controls were also enrolled in this study. Patients with RA were attending Basrah General Hospital, Rheumatology Unit, Biological Therapy Center for receiving anti-TNF therapy. Flow cytometry was used for measuring Treg/Foxp3 and Th17 markers, and the DAS-28 score was utilized to measure RA disease activity. Anti-TNF inhibitors (e.g., infliximab and etanercept), as well as other inflammatory and hematological parameters (e.g., erythrocyte sedimentation rate, total white blood cells, lymphocytes, monocytes, and neutrophil counts), were also measured in this study. DAS-28 as a disease activity score was significantly correlated with Th17/Treg/Foxp3 ratio and the Th17 cells count. Statistically, Th17/Treg/Foxp3 ratio was not correlated with body mass index, morning stiffness, and duration of the disease. Th17/Treg/Foxp3 ratio correlated significantly with DAS-28 as an RA disease activity. The lower Treg/Foxp3 frequency led to the higher DAS score reflecting higher disease activity. In the combined therapy group, disease activity was found lower than that in other patient groups indicating the effect of this combination on the relationship between MTX and anti-TNF. This study demonstrated that the main advantage of this combined therapy in RA patients was the reversion of Th17 cell expansion.