| This study was conducted for Isolation and molecular characterization of Rhodococcus equi (R. equi) isolates from human and horse specimens. A total of 203 samples (100 human sputum and 103 horses' faecal samples) were collected from different places in Baghdad, Iraq during november 2014 to april 2015. The samples were streaked on nalidixic acid-novobiocin-actidione cycloheximide-potassium tellurite (NANAT) agar, then the suspected colonies were subcultured on nutrient and blood agars. Confirmation as R equi was done by assessing the colony on the basis of their morphology and through staining and biochemical tests, followed by testing for the presence of the virulence-associated protein antigen vapA and VapB genes by multiplex PCR. Culture examination of 103 horses' faeces samples revealed the isolation of 7 (6.79%) R. equi, all of them 14.71% (7/49) were obtained from Equestrian club in Al-Ameria location and the bacteriological examination of the 100 human sputum samples showed the recovery of 4 (4.00%) isolates of R equi. The results showed that only 57.14% (4/7) of the R. equi isolates from horses' samples were vapA positive. All the 11 isolates included in this study were negative for vapB gene. This is the first study in Iraq to confirm the presence of vapA gene in R. equi isolated from horse samples indicating the contamination of the horse stables with virulent R. equi. Therefore, it is very important to take care when people deal with the animals or their environment to avoid the infection with this pathogen.