In this study, six kinds of low-carbon steel specimens with different ferrite-pearlite microstructures were fabricated by varying the Nb content and the transformation temperature. The microstructural factors of ferrite grain size, pearlite fraction, interlamellar spacing, and cementite thickness were quantitatively measured based on optical and scanning electron micrographs; then, Charpy impact tests were conducted in order to investigate the correlation of the microstructural factors with the impact toughness and the ductile-brittle transition temperature (DBTT). The microstructural analysis results showed that the Nb4 specimens had ferrite grain size smaller than that of the Nb0 specimens due to the pinning effect resulting from the formation of carbonitrides. The pearlite interlamellar spacing and the cementite thickness also decreased as the transformation temperature decreased. The Charpy impact test results indicated that the impact-absorbed energy increased and the ductile-brittle transition temperature decreased with addition of Nb content and decreasing transformation temperature, although all specimens showed ductile-brittle transition behaviour.