The nonstructural protein 5A (NS5A) of the bovine viral diarrhea virus (BVDV) is a monotopic membrane protein. This protein can anchor to the cell membrane by an in‐plane amphipathic ⍺‐helix, which participates in the viral replication complex. In this study, the effects of synonymous codon usage pattern of NS5A and the overall transfer RNA (tRNA) abundance in cells on the formation of the in‐plane membrane anchor of NS5A were analyzed, based on NS5A coding sequences of different BVDV genotypes. BVDV NS5A coding sequences represent the most potential for BVDV genotyping. Moreover, the nucleotide usage of BVDV NS5A dominates the genotype‐specific pattern of synonymous codon usage. There is an obvious relationship between synonymous codon usage bias and the spatial conformation of the in‐plane membrane anchor. Furthermore, the overall tRNA abundance profiling displays that codon positions with a high level of tRNA abundance are more than ones with a low level of tRNA abundance in the in‐plane membrane anchor, implying that high translation speed probably acts on the spatial conformation of in‐plane membrane anchor of BVDV NS5A. These results give a new opinion on the effect of codon usage bias in the formation of the in‐plane membrane anchor of BVDV NS5A.