In plant factories, the plant microclimate is affected by the control system, plant physiological activities and aerodynamic characteristics of leaves, which often leads to poor ventilation uniformity, suboptimal environmental conditions and inefficient air conditioning.  In this study, interlayer cool airflow (ILCA) was used to introduce room air into plants’ internal canopy through vent holes in cultivation boards and air layer between cultivation boards and nutrient solution surface (interlayer).  By using optimal operating parameters at a room temperature of 28°C, the ILCA system achieved similar cooling effects in the absence of a conventional air conditioning system and achieved an energy saving of 50.8% while bringing about positive microclimate change in the interlayer and nutrient solution.  This resulted in significantly reduced root growth by 41.7% without a negative influence on lettuce crop yield.  Future development in this precise microclimate control method is predicted to replace the conventional cooling (air conditioning) systems for crop production in plant factories.