The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding haemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype-specific, varying significantly in sequence and length at both the 3′ and 5′ termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3′-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multi-segment environment. These results, provide novel insights into the role of the 3′-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3′-end H1-ssNCR and observed a synergistic effect between the 3′-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of HA segment on template preference in a multi-segment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.