Tsutsui Junsai served as a bushō and, later, as a hatamoto, from the Sengoku to early Edo periods.

Junsai is deemed to have been born as the second son of Fukuzumi Toshihiro.  His mother was a daughter of Tsutsui Junshō.  Junsai is considered to have been adopted by Tsutsui Junkei.

He served Tokugawa Ieyasu and had landholdings of 5,000 koku in Fukuzumi in Yamato.  Owing to his many contributions, Junsai wed Ieyasu’s younger sister of a different mother, Ichibahime, and received landholdings of 1,000 koku in the Adachi District of Musashi Province.

In 1600, in the months prior to the Battle of Sekigahara, Junsai served in the Conquest of Aizu.  While Ieyasu’s army was in Oyama in Shimotsuke Province, news arrived that Ishida Mitsunari had raised arms.  After an impromptu war council known as the Oyama Deliberation, Ieyasu decided to forego the plan to attack Uesugi Kagekatsu and, instead, return to confront Mitsunari in Ōsaka.  At this time, Junsai was ordered to head toward Yamato and to make arrangements with Tsutsui Sadatsugu and Yagyū Muneyoshi to confirm their fidelity.

From the ninth month of 1605, he served as a guard at the western gate of Fushimi Castle.

One historical record identifies an individual named Tsutsui Kii-no-kami as a landlord in the Yamabe District of Yamato.  This appears to be in reference to Junsai.

On 8/3 of Keichō 15 (1610), Junsai died at the age of sixty.  Under another theory, he was sixty-four years old.

Junsai was succeeded by his son, Tsutsui Masatsugu.  During the Siege of Ōsaka, after a failed attempt to defend Kōriyama Castle, he took his own life.  Thereafter, the Tsutsui clan continued to serve as hatamoto of the Edo bakufu.