About 20 protesters were on the track just west of Portage la Prairie Wednesday morning until around 5 p.m. local time.
Terry Nelson, former chief of the Roseau River First Nation, said the protest was intended to get the attention of businesses that depend on CN to move their product on time.
He said protesters planned to block the rail line until Saturday.
But after Canadian National Railway (TSX:CNR) obtained an injunction against the protesters and police served it, the protesters then left.
CN spokesman Jim Feeny also says another blockade of CN's rail line in Marysville, Ont., has also ended.