Two actinobacterial strains, CPCC 203464(T) and CPCC 203448, isolated from surface-sterilized stems of medicinal plants were subjected to a polyphasic taxonomic study. These two aerobic organisms formed pale yellow colonies on tryptic soy agar (TSA). Cells were Gram-stain-positive, non-acid-fast, non-motile, rod- or coccoid-like elements. Comparative 16S rRNA gene sequence analysis indicated that strains CPCC 203464(T) and CPCC 203448 were most closely related to the type strains of the species of the genus Williamsia. Chemotaxonomic properties such as containing meso-diaminopimelic acid in the cell wall, arabinose, galactose and ribose being the whole-cell hydrolysate sugars, phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylinositol (PI) as the phospholipids, and C16 : 0, 10-methyl C18 : 0, C18 : 1ω9c, C16 : 1ω7c and/or iso-C15 : 0 2-OH as major fatty acids supported the affiliation of strains CPCC 203464(T) and CPCC 203448 to the genus Williamsia. The DNA-DNA hybridization values in combination with differentiating chemotaxonomic and physiological characteristics strongly suggested that these two isolates should be classified as representatives of a novel species of the genus Williamsia. The name Williamsia sterculiae sp. nov. is proposed, with strain CPCC 203464(T) ( = DSM 45741(T) = KCTC 29118(T)) as the type strain.