mRNAs containing premature termination codons (PTCs) are rapidly degraded through nonsense-mediated mRNA decay (NMD). However, some PTC-containing mRNAs evade NMD, and might generate mutant proteins responsible for various diseases, including cancers. Using PTC-containing human genomic β-globin constructs, we show that a fraction (~30%) of PTC-containing mRNAs expressed from NMD-competent PTC-containing constructs were as stable as their PTC-free counterparts in a steady state. These PTC-containing mRNAs were monosome-enriched and rarely contributed to expression of mutant proteins. Expression of trace amounts of mutant proteins from NMD-competent PTC-containing constructs was not affected by inhibition of eIF4E-dependent translation and such expression was dependent on a continuous influx of newly synthesized PTC-containing mRNAs, indicating that truncated mutant proteins originated primarily in the pioneer round of translation. The generation of mutant proteins was promoted by UPF1 depletion, which induced polysome association of PTC-containing mRNAs, increased eIF4E-bound PTC-containing mRNA levels, and subsequent eIF4E-dependent translation. Our findings suggest that PTC-containing mRNAs are potent and regulatable sources of mutant protein generation.