Security has been tightened at railway stations and airports across Russia.
Moscow is concerned that militants could be ramping up violence in the run-up to the 2014 winter Olympic Games in the city of Sochi in February.
The Olympics venue is close to Russia’s volatile north Caucasus region, and the BBC’s Moscow correspondent Daniel Sandford says it was always risky staging the Games so near to the troubled republics of Chechnya and Dagestan.
These bombs have been a brutal reminder of that, he says.
But a spokeswoman for the International Olympic Committee told Reuters news agency that the organisation had no doubt Russian authorities would provide adequate security for the Games.
The latest explosion took place near a busy market in Volgograd’s Dzerzhinsky district.
Maksim Akhmetov, a Russian TV reporter who was at the scene of the blast, said the trolleybus was packed with people going to work in the morning rush hour.
He described the scene as “terrible”, adding that the bus was “ravaged” and that there were “bodies everywhere, blood on the snow”.
The figures given for the number of dead and injured are still fluctuating, but investigators and the Russian health ministry told a press conference that 14 people had been killed.
At least 20 others were injured, and Health Minister Veronika Skvortsova said the patients were in “a bad condition with burns, with multiple injuries typical of blast-induced wounds”.
She said the injured include a pregnant woman, two 16 year olds and a baby aged about six months whose parents are assumed dead.
The regional governor has announced five days of mourning for all the victims.
The force of the explosion removed much of the bus’s exterior and broke windows in nearby buildings.
“It is now possible to preliminarily say that the explosive device was set off by a suicide bomber – a man whose body fragments have been collected and sent for genetic testing,” the Investigative Committee said in a statement.
City in shock
In response to this second blast in less than 24 hours, Russian President Vladimir Putin has ordered security measures to be tightened across Russia and in particular in Volgograd.
Local resident Polina Goncharova said the whole city was in shock.
“This is the first time in my life that I have experienced anything like this. I have been crying since I heard about the first bombing, and now the second one today,” she told the BBC.
“There are very few people on the streets. I am staying at home myself as I’m worried there will be more attacks.”
The first blast rocked Volgograd-1 station at around 12:45 (08:45 GMT) on Sunday, at a time of year when millions of Russians are travelling to celebrate the New Year.
A nearby security camera facing the station caught the moment of the blast, showing a bright orange flash behind the station’s main doors.
No group has yet said it was behind the blast.
Volgograd was also targeted in October, when a suspected female suicide bomber killed six people in an attack on a bus.
An Islamist insurgency in the North Caucasus region has led to many attacks there in recent years.
Insurgents have also attacked major Russian towns.
Volgograd lies about 900km (560 miles) south of Moscow, 650km north of the North Caucasus and 700km north-east of Sochi.