Background: Whether the CD40-CD40 ligand (CD40L) and inducible co-stimulatory molecule (ICOS)-ICOS ligand (ICOSL) signals are involved in chronic rhinosinusitis (CRS) development needs further investigation. Objectives: To investigate the association of CD40-CD40L and ICOS-ICOSL expression with CRS and underlying mechanisms. Methods: Immunohistology detected the expression of CD40, CD40L, ICOS, and ICOSL. Immunofluorescence was performed to evaluate the co-locations of CD40 or ICOSL with eosinophils. Correlations between CD40-CD40L and ICOS-ICOSL as well as clinical parameters were analyzed. Flow cytometry was used to explore the activation of eosinophils by CD69 expression and the CD40 and ICOSL expression on eosinophils. Results: Compared with the Non-eCRS subset, ECRS (Eosinophilic chronic rhinosinusitis) subset showed significantly increased CD40, ICOS, and ICOSL expression. The CD40, CD40L, ICOS, and ICOSL expressions were all positively correlated with eosinophil infiltration in nasal tissues. CD40 and ICOSL were mainly expressed on eosinophils. ICOS expression was significantly correlated with the expression of CD40-CD40L, while ICOSL expression was correlated with CD40 expression. ICOS-ICOSL expression positively correlated with blood eosinophils count and disease severity. rhCD40L and rhICOS significantly enhanced the activation of eosinophils from ECRS patients. TNF-α and IL-5 obviously upregulated CD40 and ICOSL expression on eosinophils, which was significantly inhibited by the p38 MAPK inhibitor. Conclusions: Increased CD40-CD40L and ICOS-ICOSL expression in nasal polyps are linked to eosinophils infiltration and disease severity of CRS. CD40-CD40L and ICOS-ICOSL signals enhance eosinophils activation of ECRS. TNF-α and IL-5 regulate eosinophils function by increasing CD40 and ICOSL expression partly via p38 MAPK activation in CRS patients.