The RSPCA is urging members of the public to come forward with any information to ascertain how the two cats, who are brothers, consumed the poison in Caerphilly, South Wales.
The devastated owner of the two cats has no idea where the poisonous substance came from.
The first - named Oliver - had to be put to sleep on veterinary advice, whilst the second - Oscar - has been undergoing treatment.
Owner Lona Grey said: "We are absolutely devastated to have lost Oliver in these circumstances, and things have been touch and go for Oscar. We have no idea how they consumed the poison.
"After our shocking ordeal, we just hope the message can get out to other local cat owners to be vigilant, and for anyone using anti-freeze to ensure that it is put away appropriately.
"I'd also like to see more information given to the general public about the dangers of anti-freeze to pets and for manufacturers to ensure their products carry a warning on packaging in the hope that events like this can be avoided."
The reports follow recent news of two fatal cat poisonings in the Pontyclun area in early November, another Caerphilly cat subject to a suspected poisoning near its Heol Y Ddol home in October, and the suspected poisoning of a white-and-grey tabby cat in Blackwood in September.
Symptoms of poisoning can include a cat vomiting, seeming depressed or sleepy, appearing drunk and uncoordinated, seizures and having difficulty breathing - and owners have been urged to be vigilant at this time.
RSPCA inspector Sophie Daniels said: "This is another hugely worrying, and sad report of a cat poisoning in the South Wales area, and our thoughts are with the owner at this time. We just hope Oscar, her other cat, can pull through.
"It is not known if the cats were poisoned deliberately or whether these incidents were a tragic accident, but given the number of recent incidents locally, it is vital cat owners are vigilant.
"Anyone who suspects their cat may have been poisoned should stay calm, remove their pet from the source of poison, contact their vet immediately, and follow their advice.
"We are deeply concerned by these reports, and would urge anyone with information to support our enquiries to contact 0300 123 8018. Calls are treated in confidence."