OBJECTIVE Immune dysregulation can affect insulin resistance (IR) and β-cell function and hence contribute to development of type 2 diabetes mellitus (T2DM). The complement system, as a regulator of immune and inflammatory homeostasis, may be a relevant contributor therein. However, longitudinal studies focusing on complement as a determinant of T2DM and IR are scarce. Therefore, we prospectively investigated the association of plasma complement factor 3 (C3) with (estimates of) IR in muscle, liver, and adipocytes, as well as with glucose tolerance, including incident T2DM. RESEARCH DESIGN AND METHODS Fasting C3, nonesterified fatty acids, glucose, and insulin (the latter two during oral glucose tolerance tests) were measured at baseline (n = 545) and after 7 years of follow-up (n = 394) in a prospective cohort study. RESULTS Over the 7-year period, C3 levels (per 0.1 g/L) were longitudinally associated with higher homeostasis model assessment of IR (HOMA2-IR; β = 15.2% [95% CI 12.9–17.6]), hepatic IR (β = 6.1% [95% CI 4.7–7.4]), adipocyte IR (β = 16.0% [95% CI 13.0–19.1]), fasting glucose (β = 1.8% [95% CI 1.2–2.4]), 2-h glucose (β = 5.2% [95% CI 3.7–6.7]), and area under the curve for glucose (β = 3.6% [95% CI 2.7–4.6]). In addition, greater changes in C3 (per 0.1 g/L) were associated with greater changes in HOMA2-IR (β = 0.08 [95% CI 0.02–0.15]) and greater changes in hepatic IR (β = 0.87 [95% CI 0.12–1.61]) over 7 years, but not glucose tolerance. Moreover, baseline C3 was associated with the 7-year incidence of T2DM (odds ratio 1.5 [95% CI 1.1–2.0]). CONCLUSIONS Changes in C3 were associated with changes in several measures of IR and may reflect progression of metabolic dysregulation, which eventually leads to abnormalities in glucose tolerance and T2DM.