Chitosan has become of interest as a crop biostimulant suitable for use in sustainable agriculture since it is biocompatible, biodegradable, environmentally friendly, and readily available in large quantity. Short-term (35 d after transplanting) effects of chitosan, applied as a soil amendment at 0%, 0.05%, 0.10%, 0.15%, 0.20%, or 0.30% (w/w), on lettuce (Lactuca sativa) growth, chlorophyll fluorescence, and gas exchange were evaluated in a growth chamber study. Chitosan at 0.05%, 0.10%, and 0.15% increased leaf area from 674 to 856, 847, and 856 cm2, and leaf fresh weight from 28.6 to 39.4, 39.1, and 39.8 g, respectively. Only chitosan at 0.05% and 0.10% increased leaf dry weight from 3.42 to 4.37 and 4.35 g, respectively, while chitosan at 0.30% decreased leaf number, area, fresh and dry weight. Chitosan at 0.10%, 0.15%, 0.20%, and 0.30% increased leaf chlorophyll index from 29.8 to 34.4, 35.4, 37.5, and 41.4, respectively. Chitosan at 0.20% and 0.30% increased leaf maximum photochemical efficiency and photochemical yield, and chitosan at 0.10%, 0.15% 0.20%, and 0.30% increased leaf electron transport rate. Leaf photosynthesis rate and stomatal conductance (gS) increased from 9.3 to 12.7, 14.0, and 16.6 μmol·m−2·s−1 carbon dioxide, and from 0.134 to 0.183, 0.196, and 0.231 mol·m−2·s−1, under chitosan at 0.15%, 0.20%, and 0.30%, respectively. The results indicated that chitosan, at appropriate application rates, enhanced lettuce growth, and might have potential to be used for sustainable production of lettuce.