The proposed project would carry oil from western Canada's tar sands to refineries on the Texas Gulf Coast.
Activists have taken their pleas to the US President, fearing the construction process will contaminate local water supplies.
"We are here for one simple reason, and it is to tell president Obama to reject the Keystone XL Pipeline in order to protect our land and water," said protest organiser, Jane Kleeb.
The 1,897 kilometre pipeline will generate more jobs and boost the economy.
But opponents to the project say it will also accelerate the effects of climate change and violate landowner rights.
Residents living on the planned route of the pipeline fear that construction will contaminate their personal water supply.
Madonna Sitting Bear is among those who oppose the plan.
"The importance of our water of the Ogallala Aquifer. We live right above it in our reservation - we come from the Rosebud Reservation - (and it) is located right above the above the aquifer in South Dakota," she said.
"That's pretty important for my son, for my nephews, for my grandchildren, you know, everybody to come; because what if it gets polluted?"
The Cowboy and Indian Alliance will continue their campaign over the next four days.
A decision is not likely to be made about the pipeline until after the US November mid-term elections.
