Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.