Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (gammaHV) infections. To completely explore the potential of the MHV-68 system for the investigation of gammaHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of gammaHV miRNAs.