After obtaining his Ph.D., Negishi decided to become an academic researcher. Although he was hoping to work at a Japanese university, he could not find a position. In 1966 he resigned from Teijin, and became a postdoctoral associate at Purdue University, working under future Nobel laureate Herbert C. Brown. From 1968 to 1972 he was an instructor at Purdue.
In 1972, he became an assistant professor at Syracuse University, where began his lifelong study of transition metal–catalyzed reactions, and was promoted to associate professor in 1979. He returned to Purdue University as a full professor in the same year.
He discovered Negishi coupling, a process which condenses organic zinc compounds and organic halides under a palladium or nickel catalyst to obtain a C-C bonded product. For this achievement, he was awarded the Nobel Prize in Chemistry in 2010. Negishi also reported that organoaluminum compounds and organic zirconium compounds can be used for cross-coupling. He did not seek a patent for this coupling technology and explained his reasoning as follows: "If we did not obtain a patent, we thought that everyone could use our results easily."
In addition, Zr(CH) obtained by reducing zirconocene dichloride is also called Negishi reagent, which can be used in oxidative cyclisation reactions. The technique he developed is estimated to be used in a quarter of all reactions in the pharmaceutical industry.
By the time Negishi retired in 2019, he had published more than 400 academic papers. He was committed to instilling rigorous practices in his lab, emphasizing the need of keeping organized and comprehensive records. Before any separations, he asked his student to evaluate crude reaction mixtures in order to minimize loss of any useful scientific information.