In the core mammalian circadian negative feedback loop, the BMAL1-CLOCK complex activates the transcription of the genes Period (Per) and Cryptochrome (Cry). To close the negative feedback loop, the PER-CRY complex interacts with the BMAL1-CLOCK complex to repress its activity. These two processes are separated temporally to ensure clock function. Here, we show that histone deacetylase 3 (HDAC3) is a critical component of the circadian negative feedback loop by regulating both the activation and repression processes in a deacetylase activity-independent manner. Genetic depletion of Hdac3 results in low-amplitude circadian rhythms and dampened E-box-driven transcription. In subjective morning, HDAC3 is required for the efficient transcriptional activation process by regulating BMAL1 stability. In subjective night, however, HDAC3 blocks FBXL3-mediated CRY1 degradation and strongly promotes BMAL1 and CRY1 association. Therefore, these two opposing but temporally separated roles of HDAC3 in the negative feedback loop provide a mechanism for robust circadian gene expression. Display omitted •HDAC3 is a critical component of the core mammalian circadian negative feedback loop•HDAC3 is required for efficient activation of E-box-driven gene expression•HDAC3 blocks CRY1 degradation and promotes BMAL1 and CRY1 association•Temporal separation of these opposing roles ensures robust circadian gene expression Shi et al. report a deacetylase-independent function of HDAC3 in maintaining a robust circadian clock. HDAC3 modulates the function of several key components of the clock machinery and affects both the activation and suppression of transcription in the circadian cycle.