Protesters, backed by senior officials, marched in silence for around 30 minutes along the National Mall near the US Capitol and Washington Monument, carrying white placards marked with the names and pictures of gun crime victims.
The demonstration came after the December shooting at Sandy Hook Elementary School in Newtown, Connecticut that saw a gunman mow down 20 young children and six adults, sparking a furious national debate over gun ownership. The shooter used a military-style assault weapon and handguns.
"No more talk. We must act, we must act, we must act," Education Secretary Arne Duncan said, telling the crowd that when he headed up a network of Chicago schools from 1990 to 2000, a child was buried every two weeks due to gun violence.
"This has to change. Our children, our families, our communities, our country deserve better," he said to thunderous applause.
Around 270 million guns are in circulation in the United States -- almost one weapon for every man, woman and child -- where more than 32,000 people were fatally gunned down in 2011 alone.
Among the protesters were several friends, neighbors and relatives of the Newtown shooting victims.
Under chilly temperatures, the demonstrators held banners reading "Ban Assault Weapons Now" and "My children are more important than your guns."
Colin Goddard, survivor of the 2007 shooting at Virginia Tech university that left 33 people dead including the gunman, said the latest spate of gun violence should trigger change.
"Enough is enough," he said. "Today is not the finish line, today is the starting block. This is not an individual race, but this is a team relay."
Washington Mayor Vincent Gray, who has come out in support of gun control, called for action to keep "each other safe from arms those irresponsible and irrational people who still go across our cities and our states and have access to guns in ways that they should not."
Duncan pledged that President Barack Obama's administration "will do everything in our power to make sure that we pass legislation that makes our children, our families, our communities safer."
In the wake of the Connecticut deaths, Obama signed 23 executive orders and also called on Congress to pass new laws in a series of sweeping measures aimed at addressing gun violence.
The proposed measures include a ban on military-style assault rifles and the closing of loopholes that allow many gun buyers to avoid background checks.
But securing congressional action will be difficult as many Republicans are vehemently opposed to the White House's plans, contending that the measures will infringe upon the constitutional right to bear arms.
Opposition to new laws is not purely along party lines. Some Democrats from states where hunting and shooting sports are popular support gun rights.
An ABC News poll this week found 53 percent of Americans back Obama's gun control plans, while 41 percent view it unfavorably.
"We know the fight will be difficult but that we will prevail," said march organizer Molly Smith.
