(1) Background: We studied how the generation position of air anions in a closed-type plant factory affects the growth characteristics of lettuce and identified the optimal position. (2) Methods: We used LEDs (red/green/blue = 8:1:1) as a light source and set the temperature and RH of the plant factory to 20 ± 2 ℃ and 50% ± 5%, respectively. We grew lettuce under three air anion conditions—sideward, upward, and downward—and compared the growth characteristics to those of a control grown without air anions. We measured the growth characteristics of the lettuce at 3 and 4 weeks after sowing, and the measurement items were shoot fresh weight (FW) and dry weight (DW); leaf area (LA), length (LL), and width (LW); SPAD; antioxidant capacity; and total phenol content. (3) Results: At 4 weeks, FW in the downward treatment condition was 25.3% higher than in the control, and DW showed a similar difference. LA was about 1943.94 cm²/plant in the downward treatment condition, which was about 15.5% higher than in the control. (4) Conclusions: We conclude that air anion generation has a positive effect on lettuce growth, and the optimal generation position for air anions is downward.