The ballot is controversial as it effectively quashed a California Supreme Court decision earlier in 2008 that had legalised gay marriage in the state.
"No More Mr Nice Gay"
Several thousand protesters rallied in front of Los Angeles City Hall on Saturday, calling for Proposition Eight to be declared invalid.
Protesters waved banners bearing slogans such as "No More Mr Nice Gay".
Anti-gay marriage activists, some carrying banners reading "Homo Sex is Sin" and "God Does Not Love You Just the Way You Are", staged a counter-protest.
A similar demonstration took place in San Diego, where around 11,000 people protested peacefully despite an attempt by anti-gay marriage activists to disrupt the march, police said.
One man was arrested for attempting to incite violence before being released.
In San Francisco, activists marched along Market Street as speakers, including local officials, urged crowds not to give up their quest for equality.
Other protests took place in New York, Chicago, Denver, Seattle, Portland, and Boston.
"Today is a national tipping point," Molly McKay, a spokeswoman for Marriage Equality USA said in a statement.
"History will look back on this day as the day that the national LGBT community rose up and said 'We are not going back'."
Meanwhile a crowd estimated at between 1,000 and 2,000 demonstrated in Salt Lake City, in Utah on Saturday, where the Mormon Church is headquartered.
The Church of Jesus Christ of Latter-day Saints, the official name of the Mormon Church, has angered gay marriage supporters after giving backing to Proposition Eight.