Two-component system AfsQ1-Q2 of Streptomyces coelicolor was identified previously for its ability to stimulate actinorhodin (ACT) and undecylprodigiosin (RED) production in Streptomyces lividans. However, disruption of either afsQ1 or afsQ2 in S. coelicolor led to no detectable changes in secondary metabolite formation or morphogenesis. In this study, we reported that, when cultivated on defined minimal medium (MM) with glutamate as the sole nitrogen source, the afsQ mutant exhibited significantly decreased ACT, RED, and calcium-dependent antibiotic (CDA) production and rapid growth of aerial mycelium. In addition, we also found that deletion of sigQ, which is located upstream of afsQ1-Q2 and encodes a putative sigma factor, led to the precocious hyperproduction of these antibiotics and delayed formation of sporulating aerial mycelium in the same glutamate-based defined MM. Reverse-transcription polymerase chain reaction and egfp fusion analyses showed that the expression of sigQ was under control by afsQ. In addition, deletion of both afsQ-sigQ resulted in the phenotype identical to that of afsQ mutant. The results suggested that afsQ1-Q2 and sigQ worked together in the regulation of both antibiotic biosynthesis and morphological development, and sigQ might be responsible for antagonizing the function of AfsQ1-Q2 in S. coelicolor, however, in a medium-dependent manner. Moreover, the study showed that the medium-dependent regulation of antibiotic biosynthesis by AfsQ1-Q2-SigQ was through pathway-specific activator genes actII-ORF4, redD, and cdaR. The study provides new insights on regulation of antibiotic biosynthesis and morphological development in S. coelicolor.