The tourists were part of a weeklong cruise that left from the Port of Long Beach on Feb. 19 and returned on Sunday.
Hooded gunmen intercepted the tourists' bus about 5 p.m. Thursday as it returned from the pueblo of El Nogalito.
The gunmen took passports, along with cameras, money, watches and other valuables before fleeing into the countryside, local papers said.
"He had his gun brandished up here, and then he brought it down and pulled up his shirt to show the knife," one woman said, recounting the incident.
"I was really scared, to be honest with you. At the same time I was a little angry because we were just minding our business, enjoying the day."
No one was injured and all the passengers returned to the ship safety.
Carnival released the following statement: "Carnival sincerely apologizes to its guests for this very unfortunate and disturbing event and is providing its full support and assistance."
The cruise line is helping victims find lost passports and other identification and is reimbursing them for any lost valuables, as well as for the trip itself.
The nature trail tour, which is operated by Carnival, has been suspended.
The Carnival Splendor usually carries more than 3,000 passengers and operates year-round weeklong cruises from Long Beach.
As of last fall, one cruise line, Princess Cruises, had decided to suspend stops in Puerto Vallarta, a popular port of call, because of the country's ongoing drug war.
Carnival itself did not cancel stops in Puerto Vallarta, and Princess Cruises was set to resume visits to Puerto Vallarta this month.
A port north of Puerto Vallarta has also experienced problems.
After a February 2011 shooting left two dead in a hotel parking lot in the tourist area of Mazatlan, several cruise lines began canceling stops, including Carnival Cruise Lines.
Spokesman Vance Gulliksen said Carnival's cancellation of visits to Mazatlan remains in effect.
"We are working with local officials as the investigation continues, and we do not have any itinerary changes as it relates to Puerto Vallarta at this time," Gulliksen said.
Earlier this month the U.S. State Department issued a travel warning to Mexico after tourism officials expressed concern over the rising crime rate in the country.