Polyomavirus family consists of a highly diverse group of small DNA viruses. The founding family member (MPyV) was first discovered in the newborn mouse in the late 1950s, which induces solid tumors in a wide variety of tissue types that are the epithelial and mesenchymal origin. Later, other family members were also isolated from a number of mammalian, avian and fish species. Some of these viruses significantly contributed to our current understanding of the fundamentals of modern biology such as transcription, replication, splicing, RNA editing, and cell transformation. After the discovery of first two human polyomaviruses (JC virus [JCV] and BK virus [BKV]) in the early 1970s, there has been a rapid expansion in the number of human polyomaviruses in recent years due to the availability of the new technologies and brought the present number to 14. Some of the human polyomaviruses cause considerably serious human diseases, including progressive multifocal leukoencephalopathy, polyomavirus-associated nephropathy, Merkel cell carcinoma, and trichodysplasia spinulosa. Emerging evidence suggests that the expression of the polyomavirus genome is more complex than previously thought. In addition to encoding universally expressed regulatory and structural proteins (LT-Ag, Sm t-Ag, VP1, VP2, and VP3), some polyomaviruses express additional virus-specific regulatory proteins and microRNAs. This review summarizes the recent advances in polyomavirus genome expression with respect to the new viral proteins and microRNAs other than the universally expressed ones. In addition, a special emphasis is devoted to the recent structural and functional discoveries in the field of polyomavirus agnoprotein which is expressed only by JCV, BKV, and simian virus 40 genomes.