Masaru Okunishi was sentenced to death in 1972 after being convicted of multiple counts of murder by slipping pesticides into wine at a community party in a remote mountain village in central Japan.
The farmer initially told police he added the toxic chemicals to kill both his wife and his mistress, so he could untangle his twisted love life. Three other women also died, while a dozen fell ill but survived.
Presiding Judge Nobuyuki Kiguchi, at Nagoya High Court, on Friday turned down Okunishi's eighth petition for a retrial, saying his defence team had failed to offer "new evidence necessary to revisit the case".
Okunishi's lead lawyer, Izumi Suzuki, said the verdict was "extremely unfortunate," and insisted the defence would file a special appeal with the supreme court.
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Okunishi, who spent decades in solitary confinement and is now hospitalised and bedridden, was acquitted at his initial trial in 1964 for lack of evidence.
Okunishi is one of the longest-serving death-row inmates in Japan, where condemned inmates can wait years or even decades in solitary confinement before being executed.
Prisoners are usually told of their impending hanging just hours beforehand, a practice critics denounce as cruel.
They also point out that Japan's justice system is far from perfect and has seen a number of high-profile miscarriages of justice.
In March last year, Iwao Hakamada was released from jail, aged 78, after decades on death row for a multiple murder he did not commit.
Apart from the United States, Japan is the only major industrialised democracy to carry out capital punishment, a practice that has led to repeated protests from European governments and human rights groups, but which enjoys public support at home.