The 5′ UTR is widely involved in gene expression via post-transcriptional regulation. However, a detailed profile of the 5′ UTR for Toxoplasma gondii has not yet been demonstrated. To investigate the issue, we compared the predicted open reading frames (ORFs) and transcription start sites (TSSs) of T. gondii obtained by TSS-seq, a method that enables analysis of encyclopedic TSSs with next-generation sequencers. As a result, it was demonstrated that the mode length of the 5′ UTR is between 120 and 140 nucleotides (nts) when a subset of genes with predicted signal peptides was examined. However, when genes without the signal peptide were examined, the length was extended to approximately 600 nts. Because additional information on the predicted signal peptide generates increased reliability to the 5′ end estimation of each ORF, we believe that the former value was more reliable as a representative of the 5′ UTR length of T. gondii. The discrepancy suggests that current predictions of the 5′ end of the ORF were less accurate and considerably more discordant with the natural status. The 5′ untranslated region (5′ UTR) is defined as that between the 5′ end of the transcripts and just in front of a start codon of an ORF. Therefore, the 5′ UTR does not contain any information for a protein sequence; however, it is involved in the control of protein expression via the modulation of translational efficiency (Kozak, 1991b; Hughes, 2006).