N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O4-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O4-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O4-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells.