Abstract: AIM　To develop an immunoconjugate with targeting antitumor effects by linking the Fab′ fragment of McAb to pingyangmycin (PYM). METHODS　McAb 3A5 was digested with pepsin to obtain Fab′ fragment. Linking between Fab′ and PYM was mediated by dextran T-40. Immunoreactivity of Fab′ was determined by ELISA. Bacteria inhibitory activity of the conjugate was determined by TTC assay. Cytotoxicity to carcinoma cells was determined by MTT assay and antitumor effects in vivo were assessed in BALB/c mice transplanted with colon carcinoma 26(C26). RESULTS　The Fab′-PYM conjugate retained immunoreactivity with C26 cells, the target cells. The IC₅₀ values of Fab′-PYM and PYM to C26 cells were 2.51　μg.mL^-1 and 32.46 μg.mL^-1, respectively. Fab′-PYM and PYM displayed similar cytotoxicity to KB cells，the non-target cells. By intraperitoneal dose of 5 mg.kg^-1×6, Fab′-PYM and PYM suppressed the growth of colon carcinoma 26 by 77% and 56%, respectively. By intravenous dose of 5 mg.kg^-1×7, Fab′-PYM, 3A5-PYM and PYM inhibited the growth of colon carcinoma 26 by 89%, 73% and 70%, respectively. Fab′-PYM was found to be more effective against target tumor than 3A5-PYM and PYM (P<0.01). With higher tolerable dose of 10 mg.kg^-1×7, iv, the inhibition of tumor growth by Fab′-PYM reached 91%. CONCLUSION　Fab′-PYM conjugate characterized by smaller size of the molecule displayed higher antitumor effects than 3A5-PYM and free PYM.