There is an ongoing discussion regarding the impact of adjuvant chemotherapy in Stage II colon cancer. We therefore estimated adjuvant treatment effect in Stage II colon cancer using pooled disease-free survival (DFS) data from randomized clinical trials (RCT approach) and compared this to real-world data (RWD approach) estimates. First, we estimated the treatment effect in RCTs by (i) searching relevant trials reporting DFS data, (ii) generating patient-level data from reported DFS data and (iii) estimating treatment effect in the patient-level data. Second, the treatment effect was estimated in an observational cohort of 1,947 patients provided by the Netherlands Cancer Registry using three propensity score methods; matching, weighting and stratification. In the RCT approach, patient-level data of 4,489 patients (events: 853) were generated from seven trials which compared two of the following treatment arms: control, 5FU/LV or FOLFOX. A Cox model was used to estimate a hazard ratio (HR) of 0.77 (0.43;1.10) for 5FU/LV vs. control and 0.93 (0.72;1.15) for FOLFOX vs. 5FU/LV. In the RWD approach, HRs for any adjuvant treatment vs. control were 0.95 (0.50;1.80), 0.88 (0.24;3.21) and 1.05 (0.04;2.06) using matching, weighting and stratification, respectively. There was no significant difference with the estimates from the RCT approach (interaction test, p > 0.10). The RCT data suggest a clinically relevant benefit of adjuvant chemotherapy in terms of DFS, but the estimate did not reach statistical significance. Stratified analyses are required to evaluate whether treatment effect differs in specific subgroups.