143 tarantulas were seized at the airport in the Colombian capital Bogotá during a raid against illegal animal trade.
The spiders were found in plastic bags on board a cargo plane, twelve were suffocated, the environment ministry said on Thursday.
The destination of the illegal delivery was Mexico.
The necessary papers for legal transport and the origin of the tarantulas were not available.
The police launched investigations to find the sender and recipient, the ministry said.
In November, 232 tarantulas destined for Europe were seized.
Colombia is one of the countries with the greatest biodiversity.
The National Animal and Environmental Protection Act provides for severe penalties for animal smuggling.
According to the International Union for Conservation of Nature (UICN), tarantulas are popular with spider enthusiasts for their tameness and colors.