The lions, both 21 years old, were euthanized Thursday due to declining health and age-related illnesses that diminished their quality of life, the zoo said in a statement.
Hubert was born at Chicago's Lincoln Park Zoo and Kalisa was born at Seattle's Woodland Park Zoo, where the pair were introduced.
They arrived at the L.A. Zoo in 2014.
Hubert fathered 10 cubs before he was paired with Kalisa, but they did not produce cubs together.
"In the early mornings, staff would routinely hear Hubert's waking roars, and I will personally miss hearing them on my walks around the grounds," Alisa Behar, curator of mammals at the L.A. Zoo, said in a statement.
"You cannot think of Hubert without thinking of his companion, Kalisa; they've been an inseparable couple for years," Behar added. "I have to commend our animal care and veterinary staff for the great care they've given this pair, a couple who lived longer than most lions do in human care and the wild."
The L.A. Zoo says the pair lived beyond the average life expectancy for African lions, which in the wild is mid-teens and about 17 years in zoos.
"Hubert and Kalisa are an iconic part of the L.A. Zoo experience, and our staff and guests have been touched by their loyal companionship," said Denise Verret, CEO & Zoo Director of the L.A. Zoo.
The African lion is native to the savannas, arid woodlands, and semi-desert regions from south of the Sahara Desert to South Africa.
African lions are categorized by the IUCN Red List as vulnerable due to human-wildlife conflict, prey depletion, the illegal trade of lion body parts for traditional medicine, trophy hunting, and disease.