Abstract: Medium-chain acyl coenzyme A synthetase (MACS) family is a subfamily of adenylate-forming enzymes superfamily, catalyzing the medium-chain fatty acids with CoA to produce medium-chain-acyl-CoA. PtMACS1 (gene model:estExt_fgenesh4_pg.c_640066) was cloned via blast in the database JGI of Populus trichocarpa. Sequence analysis showed that the conserved domains BoxI and BoxII in 4CL were not conserved in the proteins. Expression vector PtMACS1-pET-30a(+) was constructed and transformed into E. coli BL21 (DE3) to express the recombinant protein. The recombinant protein was purified by Ni-NTA affinity chromatography, enzymatic analysis showed that the recombinant protein had remarkable catalytic activity to the medium-chain fatty acids, such as hexanoic acid, nonanoic acid and decylic acid, and the Kcat were 130, 193 and 201 mol/L/(minmg) respectively. When decylic acid was used as the substrate, the optimum temperature was 37 ℃ and pH was 7.0. The results demonstrate that PtMACS1 is one of the MACS family members, and supplies research data in identifying and classifying members from adenylate-forming enzymes superfamily in Populus trichocarpa.