Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.