The blast hit the mosque in Shikarpur in Sindh province, around 470km north of Karachi, as hundreds of worshippers attended Friday prayers.
Pakistan has suffered a rising tide of sectarian violence in recent years, most of it perpetrated by hardline Sunni Muslim groups against minority Shi'ite Muslims, who make up around one in five of the population.
Sindh health minister Jam Mehtab Daher told AFP that "the death toll from the attack has increased to 61".
There are 54 dead bodies in Shikarpur hospital. Seven others died in Sukkur and Larkana hospitals," he said.
Shaukat Ali Memon, the medical superintendent of Civil Hospital in Shikarpur, earlier gave a death toll of 48.
Hundreds of people rushed to the scene after the blast to try to dig out survivors trapped under the roof of the mosque, which collapsed in the explosion, witness Zahid Noon said.
Television footage of the aftermath showed chaotic rescue scenes as people piled the wounded into cars, motorbikes and rickshaws to take them for treatment.
"The area is scattered with blood and flesh and it smells of burnt meat, people are screaming at each other... it is chaos," Noon told AFP.
Sainrakhio Mirani, police chief of the region told AFP officers were still working to determine whether it was a suicide bombing or whether the bomb was detonated remotely.
It is the bloodiest single sectarian attack in Pakistan since March 2013, when a car bomb in a Shiite neighbourhood of Karachi killed 45.
A spokesman for the shadowy Jandullah militant group, a splinter faction of the Pakistani Taliban, said they were behind the blast.
"We claim responsibility for attack on Shi'ites in Shikarpur very happily," Ahmed Marwat told AFP.
Mohabbat Ali Bablani, a Shikarpur local, said four of his cousins, aged between 30 and 40, were killed in the blast while his friend had lost five children, all under 13.
"My friend Nizam-ud-Din Sheikh has lost his five sons. He had taken them with him to offer prayers and all of them were killed in the attack," he told AFP.
Around 1000 Shi'ites have been killed in the past two years in Pakistan, with many of the attacks claimed by the hardline Sunni group Lashkar-e-Jhangvi.
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