Xer-mediated site-specific recombination plays a critical role in the segregation of circular chromosome after DNA replication. It has been shown that two tyrosine-recombinases, XerC and XerD, form a complex that resolves chromosome dimers in Escherichia coli. XerC-XerD recombinases act at a specific DNA sequence, called dif, with the aid of the ATP-dependent DNA translocase FtsK. In contrast, only a single orf (referred to as TacXerA) sharing similarity with the common Xer recombinases has been found in the genome of the archaeon Thermoplasma acidophilum, with no apparent FtsK homologue existing in this organism. Here, we provide evidence that TacXerA is a critical recombinase that resolves chromosome dimers in Thermoplasma acidophilum. ChIP-Seq analysis with anti-TacXerA antibody identified two TacXerA binding sites (named as dif1 and dif2) on the chromosome; this findig was confirmed by real-time qPCR. Consistently, in vitro experiments revealed that a dimer plasmid containing dif2 was resolved by prified TacXerA into two monomer plasmids. The catalytically important nucleotide sequence were identified by an in vitro inter-molecule recombination assay with plasmids containing various mutations in the dif2 sequence. These studies strongly suggest that TacXerA is indeed a functional Xer recombinase involved in dimer resolution of the chromosome at the dif2 site. In addition, half-site strand transfer assays with the dif2 sequence and its mutants provided further insights into how TacXerA recognizes the cleavage site in the target sequence and mediates site-specific recombination at the dif2 site.