To our knowledge, the periprosthetic joint infection (PJI) caused by Mycoplasma hominis is a rare postoperative complication after total knee arthroplasty (TKA). Just a few cases associated with Mycoplasma hominis infection after TKA were reported all over the world currently. In view of the difficulty involved in isolating this microorganism, Mycoplasma hominis infection may be under-recognized and should be considered in culture-negative cases where the microbial etiology is not easily identified. Metagenomic sequencing is a new tool to identify pathogens undetected by conventional methods. In this report, we present a case where metagenomic sequencing was used to identify Mycoplasma hominis as a novel PJI pathogen after TKA in our hospital.