The initial step in Long Interspersed Element-1 (LINE-1) retrotransposition requires transcription from an internal promoter located within its 5'-untranslated region (5'-UTR). Previous studies have identified a YY1 (Yin Yang 1)-binding site as an important sequence in LINE-1 transcription. Here, we demonstrate that mutations in the YY1-binding site have only minor effects on transcription activation of the full-length 5'-UTR and LINE-1 mobility in a single round cultured cell retrotransposition assay. Instead, these mutations disrupt proper initiation of transcription from the +1 site of the 5'-UTR. Thus, we propose that the YY1-binding site functions as a component of the LINE-1 core promoter to direct accurate transcription initiation. Indeed, this sequence may explain the evolutionary success of LINE-1 by enabling full-length retrotransposed copies to undergo autonomous retrotransposition in subsequent generations.