Abstract: To evaluate the sensitivity of Colletotrichum acutatum species complex to pyraclostrobin, delay the development of resistance and reduce the financial cost of controlling chili anthracnose, 175 single-spore isolates of C. acutatum species complex were collected from main chili production regions in Shandong Province. The sensitivities of C. acutatum species complex isolates to pyraclostrobin were determined by using mycelial growth test and spore germination test. At the same time, the joint-toxicities of pyraclostrobin combined with thiophanate-methyl, carbendazim, mancozeb, tebuconazole and fludioxonil against C. acutatum species complex were also assessed by using mycelial growth test and spore germination test. The frequency distributions of EC50 values were unimodal curves, with the average EC50 values of (0.273 ± 0.067) and (0.014 ± 0.019) μg/mL for the inhibition of mycelial growth and spore germination, respectively. Therefore, the data could be employed as the baseline-sensitivity of C. acutatum species complex to pyraclostrobin, and it could be used for the field resistance monitoring. The mean EC50 values were different among the isolates collected from different districts. The isolates from Heze were the most sensitive to pyraclostrobin, while the isolates from Weifang were the least sensitive to pyraclostrobin. The results of joint-action test indicate that the mixtures of pyraclostrobin and thiophanate-methyl at the ratios of 1:3 and 3:1 had synergistic effect on both the mycelial growth and spore germination of C. acutatum species complex. The most obvious synergistic effects were observed for the mixture of pyraclostrobin and thiophanate-methyl at the ratio of 1:3, with synergistic ratios of 4.82 and 3.94 for mycelial growth and spore germination tests, respectively. The results provided a theoretical basis for the long-term reasonable application of pyraclostrobin.