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Circulating antigen carbohydrate 125 (CA125) has emerged as a proxy of fluid overload in heart failure. This study aimed to evaluate the effect of dapagliflozin on short-term CA125 levels in patients with stable heart failure with reduced ejection fraction (HFrEF) and whether these changes mediated the effects on peak oxygen consumption (peakVO
2
). This study is a post-hoc sub-analysis of a randomized, double-blinded clinical trial in which 90 stable patients with HFrEF were randomly assigned to receive either dapagliflozin or placebo to evaluate change in peakVO
2
(NCT04197635). We used linear mixed regression analysis to compare changes in the natural logarithm of CA125 (logCA125) and percent changes from baseline (Δ%CA125). We used the “rwrmed” package to perform mediation analyses. CA125 was available in 87 patients (96.7%). LogCA125 significantly decreased in patients on treatment with dapagliflozin [1-month: Δ − 0.18, (CI 95% = − 0.33 to − 0.22) and 3-month: Δ − 0.23, (CI 95% = − 0.38 to − 0.07); omnibus p-value = 0.012]. Δ%CA125 decreased by 18.4% and 31.4% at 1 and 3-month, respectively (omnibus p-value = 0.026). Changes in logCA125 mediated the effect on peakVO
2
by 20.4% at 1 month (p < 0.001). We did not find significant changes for natural logarithm of NTproBNP (logNT-proBNP) [1-month: Δ − 0.03, (CI 95% = − 0.23 to 0.17; p = 0.794), and 3-month: Δ 0.73, (CI 95% = − 0.13 to 0.28; p-value 0.489), omnibus p-value = 0.567]. In conclusion, in patients with stable HFrEF, dapagliflozin resulted in a significant reduction in CA125. Dapagliflozin was not associated with short-term changes in natriuretic peptides. These changes mediated the effects on peakVO
2.