In the influenza virus life cycle, viral RNA (vRNA) transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the viral RNA-dependent RNA polymerase in the host cell nucleus, are delicately controlled, and the levels of the three viral RNA species display very distinct synthesis dynamics. However, the underlying mechanisms remain elusive. Here, we demonstrate that in the context of virus infection with cycloheximide treatment, the expression of viral nonstructural protein 1 (NS1) can stimulate primary transcription, while the expression of viral NS2 inhibits primary transcription. It is known that the NS1 and NS2 proteins are expressed with different timings from unspliced and spliced mRNAs of the viral NS segment. We then simulated the synthesis dynamics of NS1 and NS2 proteins during infection by dose-dependent transfection experiments in ribonucleoprotein (RNP) reconstitution systems. We found that the early-expressed NS1 protein can stimulate viral mRNA synthesis, while the late-expressed NS2 protein can inhibit mRNA synthesis but can promote vRNA synthesis in a manner highly consistent with the dynamic changes in mRNA/vRNA in the virus life cycle. Furthermore, we observed that the coexistence of sufficient NS1 and NS2, close to the status of the NS1 and NS2 levels in the late stage of infection, could boost vRNA synthesis to the highest efficiency. We also identified key functional amino acids of NS1 and NS2 involved in these regulations. Together, we propose that the stoichiometric changes in the viral NS1 and NS2 proteins during infection are responsible for the fine regulation of viral RNA transcription and replication. IMPORTANCE In order to ensure efficient multiplication, influenza virus transcribes and replicates its segmented, negative-sense viral RNA genome in highly ordered dynamics across the virus life cycle. How the virus achieves such regulation remains poorly understood. Here, we demonstrate that the stoichiometric changes in the viral NS1 and NS2 proteins during infection could be responsible for the fine regulation of the distinct dynamics of viral RNA transcription and replication. We thus propose a fundamental mechanism exploited by influenza virus to dynamically regulate the synthesis of its viral RNA through the delicate control of viral NS1 and NS2 protein expression.