The extent of the vertical microclimate heterogeneity inside a greenhouse is mostly unknown, and it can strongly affect plant production and yield quality. Tomato crop was grown in a semi-closed greenhouse equipped with horizontal ventilation and sidewall curtains, which were only opened depending on microclimate conditions; and a naturally ventilated greenhouse equipped with sidewalls curtains that were kept open. Both greenhouses had a 1,000-m2 area and a net size of 50-mesh, and were located in an arid climate zone in Israel. Vertical profiles of CO2 concentration, actual vapor pressure, air, leaf and soil temperature, net CO2 assimilation rates, stomatal conductance, and total fruit yield, fresh mass, and quality were monitored in both greenhouses for 13 days, in January 2018; CO2 concentration, actual vapor pressure, and air and soil temperature were additionally monitored in the semi-closed greenhouse for seven days in December 2016, when the ventilation was inoperative, and in December 2017, with ventilation. The vertical air temperature gradient, along with the colder microclimate inside the naturally ventilated greenhouse, led to a lack of plant uniformity and yield loss. Closing the side curtains in the fanned semi-closed greenhouse had a beneficial effect on yield, however, with mixed results for quality, due to the higher air temperature and lower carbon dioxide levels at the upper canopy. Horizontal air circulation in the semi-closed greenhouse increased transpiration and assimilation, and increased dew occurrence at night, but did not reduce the vertical heterogeneity. Significant vertical gradients affect plant physiology, and closing the curtains in winter cultivation in semi-arid/arid climates has the potential to improve fruit yield and quality. However, it must be coupled with proper air circulation and, preferably, with CO2 enrichment, or careful management of natural ventilation through side curtains, in order to maximize CO2 replenishment while minimizing heat losses.