Transcriptional regulation of primary and secondary metabolism is well-studied in Streptomyces coelicolor, a model organism for antibiotic production and cell differentiation. In contrast, little is known about post-transcriptional regulation and the potential functions of small non-coding RNAs (sRNAs) in this Gram-positive, GC-rich soil bacterium. Here, we report the identification and characterization of scr5239, an sRNA highly conserved in the genus Streptomyces. The sRNA is 159 nt long, composed of five stem-loops, and encoded in the intergenic region between SCO5238 and SCO5239. scr5239 expression is constitutive under several stress and growth conditions but dependent on the nitrogen supply. scr5239 decreases the production of the antibiotic actinorhodin, and represses expression of the extracellular agarase dagA at the post-transcriptional level by direct base pairing to the coding region 33 nt downstream of the ribosome-binding site.