Protothecaspecies are the only microalgae known to cause opportunistic infections in vertebrates and humans. Most cases of protothecosis in humans are caused byPrototheca wickerhamii, but knowledge of the pathogenicity and biology ofProtothecais limited. Globally, the diagnostic rate ofProtothecaspecies infection is much lower than the actual rate ofP. wickerhamii. The precise mechanisms underlying the pathogenesis ofProtothecainfections remain unclear. In this study, we identified a strain ofP. wickerhamiiwith atypical colony morphology. To reveal the morphological differences betweenP. wickerhamiiS1 (mucous) and the molecular basis of its pathogenicity, the transcriptomics, proteomics, and metabolomics of two pathogenicP. wickerhamiistrains and one environmental strain were analysed. Interestingly, mannan endo-1,4-β-mannosidase was significantly downregulated inP. wickerhamiiS1, which contributes to a thinner cell wall in S1 compared to strains with typical colony morphology, and the toxicity of macrophages is reduced. Metabolite analysis revealed that the mucoid appearance ofP. wickerhamiiS1 may have been caused by an increase in linoleic acid, glycerol, and other metabolites. There is still a need to better understand the ecology, aetiology, and pathogenesis ofP. wickerhamii, and in particular, its transmission between humans, animals, and the environment, from a One Health perspective.