Biobutanol (i.e., n-butanol) can be used as a substitute for spark ignition and homogeneous charge compression ignition (HCCI) engines. Its low-temperature reactions will determine the autoignition process and will have a significant effect on the heat release rate in the HCCI engines. In order to understand the temporal evolution of such reactions, the formation and concentration of intermediates formed in the oxidation of n-butanol was studied by means of a fast gas sampling probe in a single-cylinder four-stroke HCCI engine. The species in the sampled gas were identified through gas chromatography. Isooctane was used as a reference fuel to find the effect of fuel structure on the low-temperature reactions. The dominant reaction routes related to the formation of intermediate products and autoignition for n-butanol and isooctane were investigated through sensitivity analysis. The results show that for the air-fuel mixture diluted by residual gases in the cylinder, n-butanol autoignites more easily than isooctane at low temperature. For n-butanol and isooctane, the concentration of each species left in the residual gases during the negative valve overlap period is much lower than the peak concentration of its counterpart during the compression stroke. The formation of a large amount of isobutene in the cylinder before autoignition for isooctane is the cause for its late autoignition. The dominant reaction routes affecting the formation of species and autoignition timing are dependent on fuel structure.