In-situ observation of the transformation behavior of acicular ferrite in high-strength low-alloy steel using confocal laser scanning microscopy was discussed in terms of nucleation and growth. It is found that acicular ferrite nucleated at dislocations and slip bands in deformed austenite grains introduced by hot deformation in the non-recrystallization austenite region, and then proceeded to grow into an austenite grain boundary. According to an ex-situ EBSD analysis, acicular ferrite had an irregular shape morphology, finer grains with sub-grain boundaries, and higher strain values than those of polygonal ferrite. The fraction of acicular ferrite was affected by the deformation condition and increased with increasing the amount of hot deformation in the non-recrystallization austenite region.