Prolonged exposure of plants to a sufficient level of atmospheric nitrogen dioxide (NO2) activates the uptake and metabolism of nutrients that fuel plant growth and development, a phenomenon termed the plant vitalization effect of NO2. This study examined the effect of NO2 on the fruit yield of tomato (Solanum lycopersicum L. cv. Micro-Tom). Double exposure chambers, designated ±NO2 chambers, were placed in a confined greenhouse. The air entering the −NO2 chamber was scrubbed of nitrogen oxides including NO2, and the air entering the +NO2 chamber was supplemented with NO2 (50 ± 10 ppb). Two-week-old tomato seedlings that had been grown in the −NO2 chamber after sowing were then grown in a +NO2 or −NO2 chamber for the remainder of the growth period until 96 days after sowing. Growth in the +NO2 chamber led to a significant increase in fruit yield of approximately 40% as compared to growth in the −NO2 chamber. This increase in fruit yield was accompanied by acceleration of flowering time by 3.2 days and an increase in flower number per plant of up to 60%. These results show that exposure to NO2 increases fruit yield via stimulation of flowering in tomato.