Rheumatoid arthritis (RA) a chronic auto-immune condition. is characterized by inflammation that contributes to the deterioration of joints, bones, tendons and ligaments. Glucose-6-phosphate isomerase (G6PI) is one of the autoantigens against, which RA patients develop autoantibodies that have been found to circulate in serum and in synovial fluid of these patients. To evaluate serum Glucose 6 phosphate isomerase (G6PI) in patients with rheumatoid arthritis, and its role in disease activity and if associated with Anti-citrullinated peptide antibodies (ACPA) assessment to improve early diagnosis of rheumatoid arthritis (RA). This study was conducted in hospitals in Baghdad Medical city, Iraq, from December 2020 to March 2021. The study included 60 Iraqi patients (45 females, 15 males) with RA and 28 healthy subjects (18 females, 10 males) as the control group. Enzyme-linked immunosorbent assay [ELISA] used for determination of G6PI, ACPA and Abbott Architect C4000. used for the determination of RF and CRP. This study found that the levels of G6PI, ACPA, RF, CRP and ESR in patients with RA were significantly higher than in healthy controls (P-value ≤ 0.05). The study was found a positive significant correlation between G6PI and disease activity in RA patients (p-value <0.005). The study also found a positive weak significant correlation between G6PI and ACPA in rheumatoid arthritis patients (p-value ≤ 0.008) and also a statistically positive correlation significant exists between G6PI and RF in rheumatoid arthritis patients (p-value ≤ 0.05) and also a statistically positive weak significant correlation exists between G6PI and age in rheumatoid arthritis patients (p-value ≤ 0.008) in this study. In conclusion, in this study, there was significant difference in serum G6PI concentration, between patient and control group, so it seems to be a reliable marker for diagnosis of RA. The increased expression of G6PI is highly correlated with the disease activity of RA. This study showed that a positive significant weak correlation between G6PI and ACPA. May be combined with it to improve the diagnosis of RA.