We have investigated the defect effect on negative refraction of the left-handed metamaterials (LHMs). The printed circuit boards with LHMs are fabricated using a shadow mask/etching technique. The negative refraction of wedge-shaped LHMs samples with and without defects is investigated respectively. The experimental result shows that when two kinds of point defects are introduced into the sample, the ratio of the maximum power of samples with point defects to that without defects are 1.035 and 1.256, and the absolute value of the negative refraction index increases by 9.6% and 19.6%, respectively. When three kinds of vacant defects are introduced into the sample, the ratios of the maximum power of samples with vacant defects to that without defects have the highest value of 1.973 and the lowest value of 0.364, and the absolute values of the negative refraction index have increased by 68.33% and 9.6% accordingly. We think that the defect breaks the periodic structure of the sample, resulting in a new condition of the electromagnetism resonance which leads to the changes of the negative refraction index and the maximum power. So we can regulate the negative refraction index of LHMs by adjusting the defects.