In April 2009, a novel H1N1 influenza A virus (pH1N1) was recognized as the cause of the flu pandemic in humans. Here, we report the isolation of pH1N1 virus from the lung homogenates of two cats, which died after severe respiratory symptoms. The cats belonged to a cat colony consisting of 90 caged cats and were found dead following a 2-week period of respiratory and gastrointestinal diseases in the colony. During the outbreak, 25 cats died and 50% of the animal colony showed anorexia, depression, respiratory and gastrointestinal symptoms. Histological examination of the lungs of the two tested cats displayed lesions centred on terminal airways with epithelial bronchiolar hyperplasia and alveolar necrosis. Influenza A virus was detected in the lung tissues by immunohistochemistry and real-time RT-PCR (rRT-PCR). Partial sequences of haemagglutinin (HA) genes and complete sequences of neuraminidase (NA) genes of the two isolates displayed high similarity to the pH1N1 viruses circulating in humans (99% for HA gene and 100% for NA gene). To determine whether the pandemic virus had circulated among cats, serum samples and pharyngeal swabs were collected from 38 cats of the colony. Serum samples were tested by ELISA to detect antibodies against pH1N1 nucleoprotein and by hemagglutination-inhibition test, while pharyngeal swabs were examined by pH1N1 specific rRT-PCR. Twenty-one (55%) of the tested cats carried antibodies against the isolated strain and two swabs were positive for the presence of pH1N1 RNA. Our results confirm that the pH1N1 virus was able to infect cats and raise the hypothesis of the circulation of the virus within the colony being due to cat-to-cat transmission. The case reported here provides, to the best of the authors’ knowledge, the first description of the pH1N1 infection involving numerous cats that lived in a restricted area with limited contact with humans.