A series of new amphiphilic mPEG-PLA-SN38-conjugates were synthesized and formed micelles by self-assembly. Micelles of mPEG2K-PLA-SN38 have small size (∼20nm) and narrow size distribution. The lengths of mPEG and PLA chains had a major impact on the physicochemical characteristics and antitumor activity of SN38-conjugate micelles. The mPEG2K-PLA8·9k-SN38 micelles showed greater antitumor efficacy in HCT116 human colon cancer xenograft model. Display omitted •A series of amphiphilic mPEG-PLA-SN38-conjugates were synthesized and formed to micelles by self-assembly.•The lengths of mPEG and PLA chains had a major impact on the physicochemical characteristics and antitumor activity of mPEG-PLA-SN38 micelles.•mPEG2K-PLA8.9K-SN38 micelles are the most effective with complete regression of xenograft tumors. 7-Ethyl-10-hydroxy-comptothecin (SN38) is an active metabolite of irinotecan (CPT-11) and the clinical application of SN38 is limited by its hydrophobicity and instability. To address these issues, a series of novel amphiphilic mPEG-PLA-SN38-conjugates were synthesized by linking SN38 to mPEG-PLA-SA, and they could form micelles by self-assembly. The effects of mPEG-PLA composition were studied in vitro and in vivo. The mean diameters of mPEG2K-PLA-SN38 micelles and mPEG4K-PLA-SN38 micelles were 10–20nm and 120nm, respectively, and mPEG2K-PLA-SN38 micelles showed greater antitumor efficacy than mPEG4K-PLA-SN38 micelles both in vitro and in vivo. These data suggest that the lengths of mPEG and PLA chains had a major impact on the physicochemical characteristics and antitumor activity of SN38-conjugate micelles.