The newly identified influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range with broad geographical distribution. Despite the first appearance in U.S. pig herds in 2011, subsequent studies demonstrate that IDV is widespread in global cattle populations, supporting a theory that IDV utilizes bovines as a primary reservoir. Our investigation of the two reference influenza D viruses, D/swine/Oklahoma/1334/2011 (OK/11) isolated from swine and D/Bovine/Oklahoma/660/2013 (660/13) from cattle, revealed that 660/13 replicated to titers approximately 100-fold higher than those for OK/11 in multiple cell lines. By using a recently developed IDV reverse genetics system derived from low-titer OK/11, we generated recombinant chimeric OK/11 viruses in that one of the seven genome segments was replaced with its counterpart from high-titer 660/13 virus. Further characterization demonstrated that the replication level of the chimeric OK/11 virus was significantly increased only when harboring the 660/13 nucleoprotein (NP) segment. Finally, through both gain-of-function and loss-of-function experiments, we identified that one amino acid residue at position 381, located in the body domain of NP protein, was a key determinant for the replication difference between the low-titer OK/11 virus and the high-titer 660/13 virus. Taken together, our findings provide important insight into IDV replication fitness mediated by the NP protein, which should facilitate future study of infectious virus particle production mechanism of IDV. IMPORTANCE Little is known about virus infection and production mechanism for newly discovered influenza D virus (IDV) that utilizes bovines as a primary reservoir with frequent spillover to new hosts including swine. In this study, we showed that two well-characterized IDVs, 660/13 replicated more efficiently (approximately 100-fold higher) than OK/11. Using a recently developed IDV reverse genetics system, we identified viral nucleoprotein (NP) as a primary determinant of the different replication capacity observed between these two nearly identical viruses. Mechanistic investigation further revealed that a mutation at NP position 381 evidently modulated virus fitness. Taken together, these observations indicate that IDV NP protein performs a critical role in infectious virus particle production. Our study thus illustrates a NP-based mechanism for efficient IDV infection and production in vitro.