The circadian clock control of CONSTANS (CO) transcription and the light regulation of CO stability coordinately regulate photoperiodic flowering by triggering rhythmic expression of the floral integrator FLOWERING LOCUS T (FT). The diurnal pattern of CO accumulation is modulated sequentially by distinct E3 ubiquitin ligases, such as HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) in the morning, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) in late afternoon, and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) at night. In particular, CO is stabilized by FKF1 in late afternoon only under long days. Here, we show that CO abundance is not simply regulated by the E3 enzymes in a passive manner but also self-regulated actively through dynamic interactions between two CO isoforms. CO alternative splicing produces two protein variants, the full-size COa and the C-terminally truncated COb. Notably, COb, which is resistant to the E3 enzymes, induces the interactions of COa with CO-destabilizing HOS1 and COP1 but inhibits the association of COa with CO-stabilizing FKF1. These observations demonstrate that CO plays an active role in sustaining its diurnal accumulation dynamics in Arabidopsis photoperiodic flowering.