Carotenoids, particularly lutein, β-carotene, lycopene, and astaxanthin, possess established anti-inflammatory and antioxidant properties. Although these compounds are known to interact with the gut microbiota and ameliorate microbial dysbiosis, their structure-activity relationships in colitis alleviation remain poorly understood. Using a dextran sulfate sodium (DSS)-induced colitis model, we made a systematic comparison of these four structurally distinct carotenoids. All treatments markedly improved colitis-associated clinical symptoms, including weight loss, colon shortening, bloody stool, and histological damage. Notably, the fecal heme content decreased by 55.00%, 69.44%, 60.22%, and 62.24% in the lutein, β-carotene, lycopene, and astaxanthin groups by the endpoint of DSS exposure, respectively. The intervention with carotenoids significantly reduced pro-inflammatory markers while upregulating intestinal tight junction proteins and short-chain fatty acid receptor expression. 16S rRNA sequencing revealed consistent suppression of Bilophila and Mucispirillum across all groups, with structure-dependent microbiota modulation: lutein enriched Rikenellaceae, β-carotene enhanced Bifidobacteriaceae, lycopene preferentially increased Lactobacillaceae, and astaxanthin elevated Akkermansiaceae (in terms of relative abundance). These findings demonstrate that carotenoids alleviate ulcerative colitis through combined anti-inflammatory, barrier-protective, and microbiota-modulating effects, with carotenes exhibiting superior microbiota modulation compared to xanthophylls.