"Let's not waste a day on our main aim: to prepare for war and to help the people prepare for war, because it is everyone's responsibility," Mr Chavez said during his weekly radio and television show.
The comments come at a tense time for the region as Colombia signs a controversial military agreement with the United States to let US forces use seven military bases in Colombia for anti-drug operations.
Mr Chavez has repeatedly voiced deep fears of US encroachment in the region.
"Students, revolutionaries, workers, women: all are ready to defend this sacred homeland called Venezuela," he said on Sunday, adding that the best way to "avoid war is to prepare for it".
Relations between the neighbours were frozen in July after Colombia announced an agreement to lease the use of military bases to the United States.
Bilateral trade, which in 2008 exceeded $US7 billion ($7.58 billion), has largely subsided since then, notably as tensions have risen along the border
region, which extends some 2000 kilometres.
Border crossings between the countries were cut off completely this week after Caracas blamed the murder of two National Guard members near the border on "paramilitary gangs".
Venezuelan Vice President Ramon Carrizales said the pair were "assassinated by paramilitary gangs intent on taking over" the Tachira region on the border.
It is "all part of the destabilisation plan that we have been talking about for some time," Mr Carrizales, who is also the country's defence minister, said.
Mr Carrizales, however, stopped short of blaming Colombia, even though paramilitary forces are active in Venezuela's neighbour.
For years, relations have been tense between the Chavez's leftist government and the conservative government of Colombian President Alvaro Uribe, the main US ally in South America.
The death of the two Venezuelans comes after the bodies of nine Colombians were found in late October in Venezuela's Tachira state.
Mr Caracas said the dead men belonged to paramilitary forces aimed at destabilising the Chavez regime.
Mr Carrizales said in an interview with the Televen network that the shooting death of the National Guardsmen was part of a "paramilitary infiltration", part of a "systematic attack" from Colombia and the United States against Venezuela.
The 10-year deal signed in late October between Bogota and Washington, over fierce opposition from countries in the region, grants the US military access to seven military bases strategically located across Colombia.
Some 800 US troops and 600 US civilian contractors will be permitted on Colombian soil and will enjoy diplomatic immunity, which has caused an uproar among the political opposition and non-governmental organisations.
The deal, Washington says, is aimed to boost co-operation in fighting guerrillas and drug trafficking.
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