Tsutsui Junkō served as a sengoku daimyō of Yamato Province. Junkō was the grandfather of Tsutsui Junkei.
In 1484, Junkō was born as the second son of Tsutsui Junson, a bushō in the late Muromachi period and head of the Tsutsui clan who were kokujin, or provincial landowners, in Yamato. Under an alternate theory, Junkō was born on 7/7 of Meiō 2 (1493).
In 1521, Junkō succeeded his older brother, Tsutsui Junken, and became the head of the clan. He reconciled with the Ochi clan and wed his daughter to them. In the third month, after Ashikaga Yoshitane (the tenth shōgun of the Muromachi bakufu) had a falling out with Hosokawa Takakuni (the deputy shōgun), Yoshitane absconded via Izumi Province to Awaji Island and, in the tenth month, landed in the harbor town of Sakai. When Hatakeyama Hisanobu and Hatakeyama Yoshihide acted in concert with him to fight against Hatakeyama Tanenaga (Hisanobu’s son), Junkō, together with Ochi Iehide, participated on the side of Tanenaga and defeated the army of Yoshihide. Thereafter, Junkō affiliated with Tanenaga’s army and engaged in battle against Yoshihide again.
In 1528, when Yanagimoto Kataharu invaded Yamato, Junkō endured a bitter battle. Kataharu was assassinated in 1530. In the wake of his demise, in 1532, Ikkō-ikki (uprisings by members of the Ikkō sect affiliated with the Ishiyama-Hongan Temple) erupted in Yamato, whereupon Junkō collaborated with Tōichi Tōharu and the Ochi clan in an effort to suppress the uprising. This event is known as the Tenbun Disturbance.
Junkō revived the Tsutsui clan while it had been on the decline, building up their power in Yamato. He fostered marital relationships with other provincial landowners in the area such as the Tōichi clan, enabling the Tsutsui to further expand their influence.
Upon the death of Junkō in the seventh month of 1535, his lineal heir, Junshō, inherited the headship of the clan.