In 1966, he entered Grinnell College where he studied Homer's Odyssey, Dante's Inferno, constitutional history and chemistry. He married his organic chemistry lab partner, Carol Lynn Martinson, and graduated with a B.A. in 1970.

Cech's main research area is that of the process of transcription in the nucleus of cells. He studies how the genetic code of DNA is transcribed into RNA. In the 1970s, Cech had been studying the splicing of RNA in the unicellular organism Tetrahymena thermophila when he discovered that an unprocessed RNA molecule could splice itself. In 1982, Cech became the first to show that RNA molecules are not restricted to being passive carriers of genetic information – they can have catalytic functions and can participate in cellular reactions. RNA-processing reactions and protein synthesis on ribosomes in particular are catalysed by RNA. RNA enzymes are known as ribozymes and have provided a new tool for gene Technology. They also have the potential to provide new therapeutic agents – for Example, they have the ability to destroy and cleave invading, viral RNAs.

Cech's work has been recognised by many awards and prizes including: lifetime Professorship by the American Cancer Society (1987), the Louisa Gross Horwitz Prize from Columbia University (1988), the Heineken Prize of the Royal Netherlands Academy of Sciences (1988), the Albert Lasker Basic Medical Research Award (1988), the Nobel Prize in Chemistry (1989, shared with Sidney Altman) and the National Medal of Science (1995). In 1987, Cech was elected to the United States National Academy of Sciences and in 1988 he was elected to the American Academy of Arts and Sciences. In 2007, he received the Othmer Gold Medal for outstanding contributions to progress in chemistry and science.

In 1975, Cech completed his PhD in Chemistry at the University of California, Berkeley and in the same year, he entered the Massachusetts Institute of Technology where he engaged in postdoctoral research. In 1978, he obtained his first faculty position at the University of Colorado where he lectured undergraduate students in chemistry and biochemistry, and where he remains on the faculty, currently as Distinguished Professor in the Department of Chemistry and Biochemistry. In 2000, Cech succeeded Purnell Choppin as President of the Howard Hughes Medical Institute in Maryland. He also continues to head his biochemistry laboratory, the Colorado Initiative in Molecular Biotechnology, at the University of Colorado, Boulder. Cech continues to teach undergraduate chemistry at CU-Boulder. On April 1, 2008, Cech announced that he would step down as the President of HHMI, to return to teaching and research, in spring 2009.