Vestibular compensation is a natural behavioral recovery process following unilateral vestibular injury. Understanding the mechanism can considerably enhance vestibular disorder therapy and advance the adult central nervous system functional plasticity study after injury. The cerebellum, particularly the flocculonodular lobe, tightly modulates the vestibular nucleus, the center for vestibular compensation; however, it is still unclear if the flocculus on both sides is involved in vestibular compensation. Here we report that the unipolar brush cells (UBCs) in the flocculus are modulated by unilateral labyrinthectomy (UL). UBCs are excitatory interneurons targeting granule cells to provide feedforward innervation to the Purkinje cells, the primary output neurons in the cerebellum. According to the upregulated or downregulated response to the mossy fiber glutamatergic input, UBC can be classified into ON and OFF forms of UBCs. Furthermore, we discovered that the expression of marker genes of ON and OFF UBCs,mGluR1αandcalretinin, was increased and decreased, respectively, only in ipsilateral flocculus 4–8 h after UL. According to further immunostaining studies, the number of ON and OFF UBCs was not altered during UL, demonstrating that the shift in marker gene expression level in the flocculus was not caused by the transformation of cell types between UBCs and non-UBCs. These findings imply the importance of ipsilateral flocculus UBCs in the acute response of UL, and ON and OFF UBCs may be involved in vestibular compensation in opposite directions.