Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon both interact with the other three NELF subunits. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential NELF function in supporting cell growth in vitro. The majority of mouse adult tissues surveyed express the full-length NELF-B protein, and some contain a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner.