Their discoveries have unlocked insights into diseases ranging from diabetes to immune-system disorders, the Nobel Committee said.
They learned how molecules that are key to the functioning of the cell are shunted around in an internal freight system, packaged in tiny bubbles called vesicles.
They also helped resolve how the vesicles arrive on time and in the right place -- a major riddle, given that this takes place in a microscopic environment humming with movement.
If the package fails to show up at the right time, or goes to the wrong location, this can cause cellular malfunction.
"Through their discoveries, Rothman, Schekman and Suedhof have revealed the exquisitely precise control system for the transport and delivery of cellular cargo," the Nobel panel said.
Schekman told AFP he was shocked to get the call from Stockholm.
"My reaction when I heard about it was one of disbelief and joy," said the 64-year-old, a professor of molecular and cell biology at the University of California at Berkeley.
Rothman, a 62-year-old professor and chairman of the department of cell biology at Yale University, told Swedish Radio he was "profoundly honoured" to win the prize.
The trio, who are all professors at US universities, worked independently on various aspects on the vesicle system.
Schekman began his research in the 1970s, when he became interested in cellular genetics, using yeast as a model for study.
Yeast cells with defective transport machinery gave rise to a system that resembled a poorly planned public transport system, he discovered.
Then in the 1980s and 1990s, Rothman began studying vesicle transport in mammalian cells.
"He discovered that a protein complex enables vesicles to dock and fuse with their target membranes," the jury said.
The binding takes place thanks to proteins that fit with each other like the two sides of a zipper.
"The fact that they bind only in specific combinations ensures that cargo is delivered to a precise location," the citation read.
Suedhof wanted to figure out how nerve cells communicate with one another in the brain, and went on to discover in the 1990s how the vesicles' contents are released on command -- a vital part of the signaling system within the cell.
The three will share equally the prize sum of eight million Swedish kronor ($1.25 million, 925,000 euros), reduced because of the economic crisis last year from the 10 million kronor awarded since 2001.
In line with tradition, the laureates will receive their prize at a formal ceremony in Stockholm on December 10, the anniversary of prize founder Alfred Nobel's death in 1896.
Suedhof, 58, is a professor of molecular and cellular physiology at Stanford University.