Influenza A virus (IAV) is an important zoonotic pathogen, posing a severe burden for the health of both animals and humans. Many host factors are involved in the life cycle of IAV to regulate its replication. Herein, we identified sorting nexin-16 (SNX16) as a new host factor that negatively modulates the replication of IAV. When transiently overexpressed in cells, SNX16 appears to be expressed as two obvious bands. Mutagenesis analysis indicated that the amino acid residue R144 of SNX16 was responsible for its two-band expression phenotype. We found that the R144A mutation of SNX16 changed its cellular distribution in A549 cells and partially weakened the inhibitory effect of SNX16 on IAV replication. Further investigation revealed that SNX16 could negatively regulate the early stage of the replication cycle of IAV. Taken together, our results demonstrated that SNX16 is a novel restriction host factor for the replication of IAV by engaging in the early stage of IAV life cycle, and a single amino acid residue at position 144 plays an important role in the cellular distribution and anti-influenza function of SNX16.