We have previously reported that human ACAT1 mRNAs produce the 50 kDa protein using the AUG1397–1399 initiation codon, and also a minor 56 kDa isoform using the upstream in-frame GGC1274–1276 initiation codon. The GGC1274–1276 codon is located at the optional long 5′-untranslated region (5′-UTR, nt 1–1396) of the mRNAs. The DNA sequences corresponding to this 5′-UTR are located in two different chromosomes, 7 and 1. In the current work, we report that the optional long 5′-UTR significantly impairs the production of human ACAT1 protein initiated from the AUG1397–1399 codon, mainly by promoting its mRNA decay. The western blot analyses indicated that the optional long 5′-UTR potently impaired the production of different proteins initiated from the AUG1397–1399 codon, meaning that this impairing effect was not influenced by the 3′-UTR or the coding sequence of ACAT1 mRNA. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that this 5′-UTR dramatically reduced the contents of its linked mRNAs. Analyses of the protein to mRNA ratios showed that this 5′-UTR mainly decreased its mRNA stability rather than altering its translational efficiency. We next performed the plasmid transfection experiments and used actinomycin D to inhibit transcription. The results showed that this 5′-UTR promoted its mRNA decay. Additional transfection and nucleofection experiments using RNAs prepared in vitro illustrated that, in both the cytoplasm and the nucleus of cells, the optional long 5′-UTR-linked mRNAs decayed faster than those without the link. Overall, our study brings new insight to the regulation of the human ACAT1 gene expression at the post-transcription level.