Although the human neurotropic polyomavirus, JC virus (JCV), was isolated almost a half century ago, understanding the molecular mechanisms governing its biology remains highly elusive. JCV infects oligodendrocytes and astrocytes in the central nervous system (CNS) and causes a fatal brain disease known as progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals including AIDS. It has a small circular DNA genome (∼ 5 kb) and generates two primary transcripts from its early and late coding regions, producing several predicted alternatively spliced products mainly by cis‐splicing. Here, we report the discovery and characterization of two novel open reading frames (ORF1 and ORF2) associated with JCV late transcripts, generated by an unusual splicing process called trans‐splicing. These ORFs result from (i) the trans‐splicing of two different lengths of the 5'‐short coding region of VP1 between the coding regions of agnoprotein and VP2 after replacing the intron located between these two coding regions, and (ii) frame‐shifts occurring within the VP2 coding sequences terminated by a stop codon. ORF1 and ORF2 are capable of encoding 58 and 72 aa long proteins respectively and are expressed in infected cells and PML patients. Each ORF protein shares a common coding region with VP1 and has a unique coding sequence of their own. When the expression of the unique coding regions of ORFs is blocked by a stop codon insertion in the viral background, the mutant virus replicates less efficiently when compared to wild‐type, suggesting that the newly discovered ORFs play critical roles in the JCV life cycle. This article is protected by copyright. All rights reserved