The growth, cellular biochemical composition, and α-Linolenic acid content of Chlorella pyrenoidosa SHOU-1002 treated with streptomycin sulfate(STRS) at mass concentrations of 0 (CK), 20, 60, 180 and 540 μg/mL were examined. The results showed that the microalgal cell density in the CK group was significantly higher than those in STRS treated groups in the first 6 days. The biomass and single cell weight of the CK group on the 10th day were significantly higher than those of STRS treated groups. The biomass of STRS treated group decreased continuously with the increasing of STRS mass concentration in medium. The contents of Chlorophyll a, Chlorophyll b, and carotenoids in microalgae of CK, 20, 60, and 180 μg/mL STRS groups, firstly decreased then increased with prolongation of the culture time. The contents of Chlorophyll a and Chlorophyll b continuously declined while carotenoids sharply decreased in the first 4 days then stabilized in the treatments of 540 μg/mL STRS group. During the whole culture period, α-Linolenic acid was the dominant in the fatty acid profiles of C. pyrenoidosa SHOU-1002. The α-Linolenic acid contents (%) in STRS treated groups were significantly higher than that in the CK group since the 4th day. On the 10th day, the microalgae in 180 μg/mL STRS group had the highest content of α-Linolenic acid (52.09 mg/g), which increased by 108.19% as compared to that of the CK group. Moreover, the microalgae in 180 μg STRS/mL group also had the highest α-Linolenic acid yield (35.34 mg/L). The correlation analysis illustrated that α-Linolenic acid content and chlorophyll a content of the microalgae were negatively, highly, and significantly correlated. It is therefore suggested that STRS in culture medium decreased the chlorophyll a content and biomass, but promoted the accumulation of α-Linolenic acid in C. pyrenoidosa SHOU-1002.