The Democratic icon showed up for a vote on the Medicare state health program for seniors during a break from radiation and chemotherapy treatment, after undergoing surgery on a brain tumor last month.
He walked slowly from a car into the US Capitol building, then was greeted with a standing ovation from Republican and Democratic senators standing in the well of the Senate as he cast his vote.
"I return to the Senate today to keep a promise to our senior citizens and that's to protect Medicare," Kennedy, the patriach of the Kennedy political dynasty, said in a written statement.
"Win, lose or draw, I wanted to be here. I wasn't going to take the chance that my vote could make the difference."
Doctors at Duke University Medical Center in North Carolina said last month that Kennedy's brain surgery had been successful and was a first step in a treatment plan.
Kennedy was rushed to Massachusetts General Hospital in Boston on May 17 after suffering a seizure at his family's compound in Hyannis Port on Cape Cod.
Following results from a biopsy, doctors diagnosed Kennedy with a malignant glioma in the left parietal lobe, an area of the brain which controls speech, among other functions.
Doctors have not publicly offered a prognosis for Kennedy. But the US National Cancer Institute has said the outlook for such a diagnosis is poor, with average life expectancy depending on the stage of the tumor, from a few months to up to five years.
About 13,000 Americans die annually from malignant tumors in the brain or spinal cord, comprising 2.2 percent of all cancer-linked deaths, according to the American Cancer Society. Survival has improved over the past decade due in part to new drugs.
The tumor kills 50 percent of patients during the first year after diagnosis and few live beyond three years. Without treatment the tumor grows back between two to three months after being surgically removed.
The brain tumor diagnosis sent shockwaves through the US Congress, where Kennedy has been a dominant figure for nearly half a century and is a champion of causes such as health care, education, workers rights and immigration reform.
Kennedy, whose eighth six-year term in the Senate expires in 2012, once seemed destined for the White House.
But his career was rocked by the death of a young woman, Mary Jo Kopechne, in his car late one night in 1969 after he drove off a bridge near Chappaquiddick island.
He did run for president in 1980 against incumbent Jimmy Carter. Kennedy lost the Democratic nomination but politically damaged Carter, who lost the general election to Republican Ronald Reagan.
Kennedy's brother, late president John F. Kennedy Jr., was shot and killed in 1963, and brother Robert Kennedy was shot dead while campaigning for the presidency in 1968.
His eldest brother Joseph died in a plane crash during World War II.