The main symptom of boron (B) deficiency in cotton is the formation of brown rings on leaf petioles. The objective of the present study was to determine the changes in the anatomical structure and chemical composition of petioles and photosynthesis of leaves in cotton under B deficiency. Compared to the control, B deficiency treatment resulted in large increases in the number of petioles with brown rings per plant (160.0%) and the number of rings on the petiole per functional leaf (711.1%) in cotton seedlings. The relative absorbance intensity in the fingerprint region of polysaccharide structure was decreased in petiole rings under B deficiency, while lignin contents were increased. Cotton plants mitigated the impairment of transport function in cotton petioles by increasing the areas of vascular bundles, phloem, xylem, and phloem fiber. Moreover, the stomatal conductance, photosynthetic rate, and transpiration rate in leaves were significantly decreased under B deficiency, thus impeding photosynthesis in cotton plants. Therefore, B deficiency reduces transport function in petioles and photosynthesis in leaves, and leads to the formation of noticeable brown rings on petioles of cotton seedlings.