The impact of injection timings on the in-cylinder flow field intensity, mixture formation process, combustion, and emission characteristics are analyzed. The range of injection timing is set between −320°CA and −260°CA. The research results indicate: Delaying the injection timing causes the spray plumes increasingly align with the tumble direction, consequently enhancing the in-cylinder tumble intensity and the turbulence near the spark timing. The mixture homogeneity is influenced by both the formation time and the in-cylinder flow field intensity. Moreover, combustion performance is linked to the homogeneity of the mixture and turbulence intensity near the spark timing. Despite the fact that postponed injection may augment both turbulence intensity and the equivalent ratio around the spark plug at the moment of spark, achieving higher explosion pressure, but the higher viscosity of n-butanol impedes droplet break-up, and its low volatility decelerates the evaporation rate, leading to poorer mixture homogeneity, reduced BMEP, and increased CO and soot emissions. However, at −300°CA start of injection, the extended mixing time optimizes the fuel-air mixture homogeneity, thus improving combustion quality and reducing CO, soot, and HC emissions, albeit leading to increased NOx emissions.