CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3'-OH and 5'-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibited no effect on the expression of the nicked DNA in human fibroblasts immortalised by SV40 large T-antigen but caused a very strong decrease of gene expression in spontaneously transformed mouse embryonic fibroblasts (MEFs). Compared to the corresponding CSB-proficient MEFs, the effect was on average 6.7-fold stronger for a defined SSB located in the non-transcribed DNA strand, but only 2.4-fold for a SSB in the transcribed strand and 1.7-fold for a SSB located in the non-genic region. At the same time, CSB deficiency did not compromise the overall efficiency of repair of SSBs generated by treatment of the cells with hydrogen peroxide. The gene expression data thus indicate that CSB prevents irreversible transcription failures at the sites of DNA damage, acting preferentially at SSBs located in the non-transcribed DNA strand of the transcribed genes. We further conclude that SSBs in the non-transcribed DNA strand are commonly more harmful for transcription than those situated in the transcribed strand.