Recent studies have revealed that the biological activity of astaxanthin differs among E/Z-isomers. Therefore, it is essential to investigate the distribution of astaxanthin isomers in the body to comprehensively elucidate their role. However, owing to the technical complexity of astaxanthin isomer analysis, detailed information regarding the precise distribution of isomers in the body remains limited. In this study, food-grade astaxanthin esters derived from Haematococcus lacustris (total Z-isomer ratio of astaxanthin = 34.5%) were administered to male Sprague-Dawley rats, and their plasma response and tissue distribution were investigated. Astaxanthin isomers were analyzed using normal-phase high-performance liquid chromatography, which can accurately measure the isomers. The maximum plasma concentration (Cmax) and area under the curve from time 0 to 24 h (AUC0-24 h) values of total astaxanthin isomers were determined to be 20.4 ± 4.6 ng/mL (Tmax: 8.3 ± 2.3 h) and 261.8 ± 16.4 ng·h/mL, respectively. The total Z-isomer ratio of astaxanthin in the plasma increased over time and reached approximately 55% at 24 h post-administration. Astaxanthin accumulated in the liver, kidneys, lungs, and testes exhibited a high total Z-isomer ratio (> 44.0%). In plasma and tissues, the predominant astaxanthin Z-isomer was the 13Z-isomer, and minor quantities of 9Z-, 15Z-, and two unidentified astaxanthin Z-isomers (potentially multi-Z-isomers) were observed. The composition of astaxanthin isomers in the plasma and tissues differed substantially from that of the diet. These findings suggest that astaxanthin Z-isomers, particularly the 13Z-isomer, possess higher absorbability than the all-E-isomer, or that a mechanism regulates astaxanthin isomer composition in the rat body.