In 1904, Nogi was called to active service on the occasion of the Russo-Japanese War, and was promoted to army general in command of the Japanese Third Army, with an initial strength of approximately 90,000 men and assigned to the capture of the Russia port of Port Arthur on the southern tip of Liaodong Peninsula, Manchuria. Nogi's forces landed shortly after the Battle of Nanshan, in which his eldest son, serving with the Japanese Second Army, was killed. Advancing slowly down the Liaodong Peninsula, Nogi encountered unexpectedly strong resistance, and far more fortifications than he had experienced ten years earlier against the Chinese. The attack against Port Arthur quickly turned into the lengthy Siege of Port Arthur, an engagement lasting from 1 August 1904 to 2 January 1905, costing the Japanese massive losses, including Nogi's second son. Due to the mounting casualties and failure of Nogi to overcome Port Arthur's defenses, there was mounting pressure within the Japanese government and military to relieve him of command. However, in an unprecedented action, Emperor Meiji spoke out during the Supreme War Council meeting, defending Nogi and demanding that he be kept in command.
After the fall of Port Arthur, Nogi was regarded as a national hero. He led his 3rd Army against the Russian forces at the final Battle of Mukden, ending the land combat phase of operations of the war.
British historian Richard Storry noted that Nogi imposed the best of the Japanese samurai tradition on the men under his command such that "...the conduct of the Japanese during the Russo-Japanese War towards both prisoners and Chinese civilians won the respect, and indeed admiration, of the world."
At the end of the war, Nogi made a report directly to Emperor Meiji during a Gozen Kaigi. When explaining battles of the Siege of Port Arthur in detail, he broke down and wept, apologizing for the 56,000 lives lost in that campaign and asking to be allowed to kill himself in atonement. Emperor Meiji told him that suicide was unacceptable, as all responsibility for the war was due to imperial orders, and that Nogi must remain alive, at least as long as he himself lived.
Nogi and his wife committed seppuku shortly after the Emperor Meiji's funeral entourage left the palace. The ritual suicide was in accordance with the samurai practice of following one's master to death (junshi). Nogi and his spouse bathed together, and changed into white kimonos, before sharing a cup of sake before the tokonoma. Mrs. Nogi proceeded to commit suicide first; while Nogi assisted by plunging a dagger into her neck. He then sliced his own stomach open. In his suicide letter, he said that he wished to expiate for his disgrace in Kyuushuu, and for the thousands of casualties at Port Arthur. He also donated his body to medical science.
All four members of the Nogi family are buried at Aoyama Cemetery in Tokyo. Under State Shinto, Nogi was revered as a kami and a Shinto shrine in his honor still exists on the site of his house in Nogizaka, Tokyo.