CONSTANS (CO) plays a vital role in activating the expression of the florigen FT during photoperiod-dependent flowering. The diurnal oscillation of CO protein abundance is strictly controlled by daylength and the circadian clock. We showed previously that ABI5-BINDING PROTEIN2 (AFP2) represses CO expression to delay flowering. In this study, we identified DNAJ HOMOLOG 3 (J3) as an AFP2-interacting protein in Arabidopsis thaliana. The interaction between J3 and AFP2 was confirmed through a series of biochemical experiments. In long days, the j3 mutant exhibited pronounced late flowering with reduced CO protein abundance, whereas overexpressing J3 accelerated flowering time by stabilizing CO, suggesting that J3 acts as a positive regulator of photoperiod responses. Furthermore, J3 suppressed the interaction between CO and AFP2, and protected CO from AFP2-mediated degradation, thus efficiently activating FT expression at the end of the day. Consistent with these data, the late-flowering phenotype caused by overexpressing AFP2 was reversed by overexpressing J3 in planta, whereas the early-flowering phenotype of the afp2 mutant was compromised in the afp2 j3 double mutant. Together, these results reveal a novel function for J3 in coordinating CO stability through antagonizing AFP2 activity, thus fine-tuning the downstream FT expression during photoperiodic flowering.