Phosphofructokinase (PFK) is a rate‐limiting enzyme in glycolysis, but its linkage with locomotion in termites is not well understood, despite the demonstrated involvement of this gene in the locomotion of different animals. Here, we investigated the effect of the pfk gene on locomotion in the subterranean termite Reticulitermes chinensis Snyder through RNA interference and the use of an Ethovision XT tracking system. The knockdown of pfk resulted in significantly decreased expression of the pfk gene in different castes of termites. The pfk‐silenced workers displayed higher levels of glucose but lower levels of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) production and decreased activity of the PFK enzyme. Furthermore, abnormal locomotion (decreased distance travelled, velocity and acceleration but increased turn angle, angular velocity and meander) was observed in different castes of pfk‐silenced termites. We found caste‐specific locomotion among workers, soldiers and dealates. Additionally, soldiers and dealates showed higher velocity in the inner zone than in the wall zone, which is considered an effective behaviour to avoid predation. These findings reveal the close linkage between the pfk gene and locomotion in termites, which helps us to better understand the regulatory mechanism and caste specificity of social behaviours in social insects.