NSW Health has confirmed the man aged in his 70s from the Griffith region died in a Sydney hospital on February 13.
Autoposy results released on Wednesday showed the man had the mosquito-born virus.
He is the second Australian to have died from the disease. The first was a Victorian man in his 60s who died on February 28.
There are now 15 human cases of the virus in Australia, including seven in Victoria, one in Queensland, and three in NSW.
SA Health on Wednesday said four suspected cases had been confirmed to have Japanese encephalitis, all required hospitalisation and three acquired the infection locally.
NSW Health said several more people in NSW were undergoing further testing, with more cases expected over the coming days.
The disease, which spreads to humans through mosquito bites, has infected at least 20 piggeries across the country.
Victoria's Deputy Chief Health Officer Deborah Friedman said Japanese encephalitis had now been found at piggeries in the local government areas of Loddon, Campaspe, Gannawarra, Bendigo, Shepparton and Wangaratta.
She said authorities were several weeks to months behind detecting the virus, with pigs likely first exposed between September and November.
"The reason it was detected or suspected in piggeries was because of what they refer to as reproductive losses," Dr Friedman told reporters.
"Pigs were having stillborn piglets, or what they refer to as mummified piglets, which means that they've died in utero some time before."
Dr Friedman said with pig pregnancy lasting 115 days, they had to have been infected during that time, taking exposure back to late last year.
The federal government is working closely with states and territories to support the distribution of vaccine doses to at-risk population groups.
Vaccination is recommended for people who work with or around pigs, including transport workers, vets and those who cull or hunt the animals.
Victorian Health Minister Martin Foley said the virus was spreading due to climate change, following heavy rain and devastating floods in NSW and Queensland.
"Clearly with so much climate change-induced weather pattern change, we're now seeing it move around all of the states," he said.
"Whilst only about one per cent of cases display symptoms, and only a very small number of those display extreme symptoms requiring hospitalisation, it is nonetheless a nasty disease."
Japanese encephalitis cannot be transmitted from person-to-person, nor by eating pork products, with children under five and older people at highest risk of developing serious disease.
Anyone suffering from severe symptoms, including nausea, headaches, light sensitivity and fever should urgently seek medical attention.
All people, but particularly those in areas with high mosquito activity, are urged to avoid being bitten by mosquitoes by wearing repellent and covering up exposed skin.
It has been more than 20 years since Japanese encephalitis was detected in humans. The last outbreak was in Australia's tropical north in 1995.
Australia's chief medical officer last week declared Japanese encephalitis a nationally significant communicable disease incident, triggering extra resources for states and territories.