A new Amnesty report says paramilitaries are still operating and colluding with the security forces to eliminate and intimidate union members.
The Colombian authorities have taken steps to improve union members' safety, Amnesty says, and the number killed has fallen in recent years.
Nevertheless, in 2006, 77 trade unionists were killed or "disappeared".
Since 1991, some 2,245 members of trade unions have been killed, 3,400 threatened and more than 130 have "disappeared", according to figures from Colombia's National Trade Union School.
Trade unionists involved in labour disputes and campaigns against privatisation are particularly targeted, Amnesty's report says.
Also at risk are those who work in some of Colombia's key sectors, such as oil, mining, gas and energy.
"Trade unionists across Colombia are being sent a clear message: don't complain about your labour conditions or campaign to protect your rights because you will be silenced at any cost," said Susan Lee, Amnesty's Americas programme director.
Demobilisation
Thousands of paramilitary fighters have demobilised in a controversial government process over the past three years.
But Amnesty International says there is strong evidence that paramilitary groups still operate and are responsible for human rights violations including threats, killings and enforced disappearances, sometimes in collusion with the security forces.
One case detailed in the Amnesty report is that of Luciano Enrique Romero Molina, who was found dead in September 2005.
His hands had been tied and he had been stabbed more than 40 times.
His body was found in a neighbourhood of Valledupar, an area still said to be controlled by paramilitaries, despite the fact that paramilitary forces operating there were supposedly engaged in a process of demobilisation between December 2004 and March 2006.
Guerrilla forces were also blamed for some, although far fewer, deaths of trade unionists.
Amnesty notes that the Colombian authorities have taken action to improve the safety of trade unionists, including providing bodyguards, bullet-proof vehicles and mobile phones for those who have been threatened.
But the group says that sometimes these security measures have been withdrawn or curtailed, with lack of funds often used to justify this.
Amnesty is also urging companies working in Colombia to use their influence with the government to end and prevent human rights abuses.
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