California poppy products are commonly used for the treatment of nervousness, anxiety and sleeping disorders. Pharmacologically relevant constituents include the main alkaloids californidine, escholtzine and protopine. However, only limited information is available about the alkaloid content in commercial preparations and their intestinal absorption. Moreover, a possible metabolization of these alkaloids by the gut microbiota, and their impact on microbial activity and viability have not been investigated. Californidine, escholtzine and protopine were quantified by UHPLC-MS/MS in eight commercial California poppy products. The intestinal permeability of alkaloids was studied in Caco-2 cell as a model for absorption in the small intestine. The gut microbial biotransformation was explored in artificial gut microbiota from the in vitro PolyFermS model. In addition, the impact of these alkaloids and a California poppy extract on the microbial production of short-chain fatty acids (SCFAs) and the viability of microbiota was investigated. Contents of californidine, escholtzine and protopine in California poppy products were in the ranges of 0.13–2.55, 0.05–0.63 and 0.008–0.200 mg/g, respectively. In the Caco-2 cell model, californidine was low-to-moderately permeable while escholtzine and protopine were highly permeable. An active transport process was potentially involved in the transfer of the three alkaloids. The three compounds were not metabolized by the artificial gut microbiota over 24 h. Neither the California poppy extract nor the alkaloids markedly impacted microbial SCFA production and bacterial viability.