ABSTRACT: The study demonstrated the gene expression through microarray analysis of total mRNA in rams experimentally infected with a rough (R) virulent strain of Brucella ovis in reproductive organs (epididymus, testicles, ampolae, vesicular glands, bulbourethral glands) and a pool of lymph nodes (inguinal and scrotal) at three different times: acute phase (60 days post challenge [dpc]), chronic phase 1 (120dpc), and chronic phase 2 (240dpc) of infection. To further define the gene expression changes associated with infected rams, the profiles of a control group (0 dpc) of rams was compared using the Affymetrix Bovine Genome Array. Of the 23,000 genes that were analyzed on the array, B. ovis infection in rams’ tissues revealed 139, 930 and 744 Differentially Expressed Genes (DEGs) in the acute, chronic 1, and chronic phase 2 of infection, respectively. Among the three phases of infection, 44 DEGs (30 known and 14 unknown genes) were expressed in common. The biological functions of immune cell trafficking, immunological disease, infectious disease, inflammatory disease, inflammatory response and cellular movement were significant at the three phases of infection. The results support the first microarray analysis of ram tissues infected with an R strain of B. ovis. A total of twelve 12-month-old healthy Santa Inês rams, previously diagnosed as negative for brucellosis/visna maedi/toxoplasmosis/neosporosis, were selected for the study. The rams were kept in an isolated pen and experimentally challenged with a rough (R) virulent B. ovis PA strain (ATCC 25840). For the experiment, the rams were classified during the course of infection according to time: 0 dpc (control), 60 dpc (acute phase), 120 dpc (chronic phase 1) and 240 dpc (chronic phase 2). For each time, the tissues epididymus, testicles, ampolae, vesicular glands, bulbourethral glands, and a pool of lymph nodes (inguinal and scrotal) were collected. Three rams were euthanized and necropsied for tissues collection purposes for each time. To define the gene expression changes associated with infected rams, the profile of a control group (0 dpc) of rams was compared using microarray technology. REPLACE This dataset includes control samples and chronic 2 phase samples.