Rare　sugars　have　received　increasing　attention　due　to　their　important　applications　as　sweeteners　and　building　blocks.　The　substrate　specificity　and　catalytic　properties　of　ribose-5-phosphate　isomerase　A　(RpiA)　in　isomerization　of　rare　sugars　have　not　been　extensively　explored.　In　this　study,　an　RpiA　from　Ochrobactrum　sp.　CSL1　was　cloned　and　expressed　in　Escherichia　coli.　The　biochemical　and　reaction　features　were　explored　and　its　broad　substrate　specificity　was　identified.　A　higher　reaction　rate　in　isomerizing　L-rhamnose　to　L-rhamnulose　by　OsRpiA,　compared　with　OsRpiB　found　in　the　same　strain　indicated　higher　efficiency　in　preparing　rare　sugars,　which　was　verified　by　kinetics　study.　The　2.8　Å　resolution　structure　of　OsRpiA　was　then　solved　and　used　in　subsequent　molecular　dynamics　experiments,　providing　a　possible　explanation　for　its　distinct　substrate　specificity.　The　present　study　highlighted　the　unique　role　of　microbial　RpiA　in　preparing　rare　sugars,　and　its　structural　information　provided　a　reliable　reference　for　further　reaction　mechanism　research　and　enzyme　engineering　work.　©　2020　Elsevier　Inc.