The hotline is being reopened on the 27th anniversary of his shooting in central Stockholm, the Swedish news agency TT reported.
By re-establishing the tip line, police are hoping to find people "who have thoughts on the matter who never contacted us because they assumed others would have said the same thing," investigator Dag Anderson wrote on the police website.
No one has ever been permanently tagged for the murder, which occurred as Palme and his wife walked home from a late-night movie. One man was convicted, but acquitted on appeal.
The Social Democrat was leader of the workers movement in Sweden and an outspoken critic of the war in Vietnam and apartheid in South Africa.