UNLABELLED: The present study aimed to newly synthesize C-Phycocyanin nanoparticles through a ball-milling technique. C-Phycocyanin nanoparticles had average diameter of 290.2 nm, zeta potential 48.3 mV and 0.390 PDI. The synthesized nanoparticles were tested as an anti-cryptosporidiosis agent compared to the crude C-Phycocyanin. Cryptosporidiosis induction was done in immunocompetent and immunosuppressed mice. The antiparasitic effect was evaluated through multiplex qualitative PCR, count of oocysts, serum biochemical parameters, oxidative stress and antioxidant markers, cytokine analysis, and histopathological study (qualitative and quantitative). Multiplex qualitative PCR analysis revealed the presence of the tested parasite gene (JVAF) in all the treated groups. The percentage of the highest reduction of the oocysts means counting has been detected in the infected mice treated with nitazoxanide (NTZ) (Ic, IIc) followed by treatment with C-Phycocyanin nano (Ie, IIe), then C-Phycocyanin (Id, IId) (42, 48, 37, 36 15, and 29% respectively). C-Phycocyanin and C-Phycocyanin nanoparticles treated groups dramatically affected the levels of catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) activity. Moreover, treatment with C-Phycocyanin and C-Phycocyanin nanoparticles significantly reduced cytokines levels (Tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin (IL-13)) in contrast to untreated groups. The histological results in the tissues of mice's ileum which are infected by SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12639-024-01739-2).