•Excipient emulsions increase the bioaccessibility of β-carotene in spinach.•Mixed micelles promote osmotic absorption of β-carotene in the intestine.•LCT facilitates the entry of β-carotene into the circulation via the lymphatic pathway.•Excipient emulsions improve the biological activity of β-carotene in vivo. This study sought to improve the biological fate of β-carotene obtained from spinach, using in vitro digestion, in situ single-pass intestinal perfusion, and in vivo approaches, to investigate the effects of excipient emulsions with medium- (MCT) and long-chain triglyceride (LCT) as a vehicle for improved health benefits of β-carotene. Results showed that the bioavailability and bioactivity of β-carotene were both significantly higher in the excipient emulsions relative to those without the emulsions. This was especially true when LCT was used as the vehicle. These results were confirmed by bioaccessibility, duodenal absorption, and in vivo absorption and metabolism. Furthermore, animal feeding studies revealed that LCT may have the potential to promote triglyceride and apo-B48 reconstitution and secretion. This suggested that LCT may facilitate the entry of carotenoids into circulation via the lymphatic pathway. These results highlight the importance of the optimization of excipient foods to improve the efficacy of lipophilic carotenoid.