Abstract: Trichoderma harzianum Th-33 antagonize against a variety of plant pathogenic fungi. Thga1 (GenBank JN874387) of strain Th-33 codes a type I GTP binding protein α subunit. Two Thga1 gene knock-out mutants ΔThgal 1-1 and ΔThgal 10-1 had been constructed in our previous studies. The antagonistic effects of the two mutants against three plant pathogens including Rhizoctonia solani, Phytophthora capsici and Fusarium oxysporum were studied in this paper. The two mutants could not cover and mycoparasitize the colonies of tested pathogens and the antagonisms reduced significantly comparing to wild-type Th-33 when confronted with tested plant pathogens. The inhibitory ratios of volatile metabolites from ΔThgal 1-1 and ΔThgal 10-1 to the tested plant pathogens reduced 50%—57% to R. solani, 43%—58% to P. capsici and 58%—63% to F. oxysporum comparing to that of wild-type Th-33 respectively, and there was no significant difference between the two mutants. Inhibition ratios of non-volatile metabolites from ΔThgal 1-1 and ΔThgal 10-1 to the three tested plant pathogens reduced 8%—25% to R. solani, 23%—56% to P. capsici and 38%—50% to F. oxysporum, respectively. The parasitism of wild-type Th-33 against R. solani such as hyphae winding, adhering, penetration and leading to hyphae disruption were observed under microscope, but we have not observed the above phenomena resulting from the two mutants. The results showed that Thga1 influenced antagonism, secondary metabolism and mycoparasitism of T. harzianum.