The study of sperm-mycoplasma interaction has been focused on the effects of infection on sperm quality, but few studies have reported the direct interaction of this bacterium with spermatozoa.Selected populations of viable, motile and infection-free human spermatozoa from three healthy men were incubated with 15-480 multiplicity of infection (MOI) units of DiIC18-labelled Mycoplasma hominis. Cells were analyzed by means of confocal microscopy and by the eosin-Y dye exclusion test between 10 min and 24 h post-infection.As early as 10 min post-infection, clusters of M. hominis were seen attached to the sperm head, midpiece or tail. Mycoplasma showed an approximately 2.5-4.5-fold higher interaction with sperm head or tail than with midpiece. Sequential sectioning of infected spermatozoa revealed the intracellular location of M. hominis within cytosolic spaces of head and midpiece regions. A minor proportion of infected spermatozoa showed bent or coiled tails, and/or midpiece thickening. Sperm viability was not altered by M. hominis infection.These results provide specific and conclusive evidence of M. hominis attachment and invasiveness towards human sperm cells, which seems not to affect their viability, suggesting that a short-term M. hominis interaction with spermatozoa results in non-apparent or subtle damage, but might have implications for long-term male or couple's fertility.