Some roshi (masterless samurai) of the Mito and Satsuma clans resented the repeated crackdowns of the bakufu government and started to plan an assassination, and on March 24, 1860, successfully attacked and killed Ii, who was about to enter Edo-jo Castle, in the outer field of the Sakurada-mon Gate (the Sakuradamon Incident).
度重なる弾圧に憤慨した水戸藩や薩摩藩の浪士は、密かに暗殺計画を練り、万延元年3月3日(旧暦)（1860年3月24日）、江戸城登城の途中の井伊を桜田門の外で襲撃して暗殺を決行した（桜田門外の変）。