Abstract: Three medium-carbon vanadium-microalloyed steels were designed and experimentally prepared, with carbon contents of 0.26%, 0.34% and 0.42%. The effect of carbon content on the formation of intragranular ferrite (IGF) in the steels isothermally treated at 600, 550, 500 and 450℃ were studied. Vickers hardness test was carried out for the steels isothermally treated at different temperatures. The nano-hardness of grain boundary ferrite (GBF) and IGF was also measured. Nucleation sites of IGF were then discussed. The results showed that the amount of non-eutectoid ferrite increased, the grain size of ferrite decreased, and the hardness was enhanced with increasing carbon content, if isothermally treated at the same temperature. In samples isothermally treated at 500℃, the nano-hardness of GBF was found higher than that of IGF. The nano-hardness of both GBF and IGF increased with the increase of carbon content, but IGF's varied little. It was also found that appropriate inclusions and pre-nucleated IGF could be nucleation sites of IGF.