CD40 activation on dendritic cells (DCs) enhances tumor antigen cross-priming of tumor-specific cytotoxic T lymphocytes, strengthening anticancer immune responses. RO7300490 is a fibroblast activation protein (FAP)-targeted CD40 agonist antibody. In this phase I study, 80 patients with advanced and/or metastatic solid tumors received RO7300490 biweekly (dose range 16-1,100 mg). The primary objective was to evaluate safety and tolerability. Secondary/exploratory objectives included pharmacokinetics, antitumor activity and pharmacodynamics. Treatment-related adverse events (TRAEs) occurred in 53 patients (66.3%) and were mostly grade 1-2. Grade 3-4 TRAEs (3.8%) and TRAEs leading to discontinuation (2.5%) were uncommon. No grade 5 TRAEs were reported. RO7300490 showed target-mediated drug disposition, with sustained exposure at higher doses. No objective responses and limited clinical activity (disease control rate 42.5%) were observed despite rapid and persistent tumor uptake of radiolabeled RO7300490. Intratumoral pharmacodynamic activity was demonstrated by a significant increase in DC-LAMP+ DC density in paired tumor biopsies. An increase in B cell density was also observed, along with the formation of pretertiary lymphoid structures, co-organized in focal micro-neighborhoods with DCs. In summary, treatment with a tumor-targeted CD40 agonist antibody is feasible, clinically manageable and induces immunomodulation of the tumor microenvironment. ClinicalTrials.gov registration: NCT04857138 .