The genus Colletotrichum includes a number of important plant pathogens, which cause anthracnose diseases on a broad range of hosts in the world. In recent years, walnut has been severely damaged by anthracnose disease in China with significant yield losses. Thus, it is necessary to verify the etiology of anthracnose on walnut using both morphological and molecular approaches. In 2014, walnut fruits with anthracnose symptoms were collected from five walnut orchards in Shandong Province, China, and 24 isolates were isolated. Among them, six similar single-spore isolates obtained were used for pathogenicity testing of walnut anthracnose. Acervuli were brown, circular and the average size was 50.4–101.8 μm. Conidiophores were hyaline, septate, not branched or branching at the base, conidiogenous cells were enteroblastic, phialidic, hyaline, cylindrical, ampulliform. Conidia were single celled, smooth-walled with a large guttule, colorless, fusiform to cylindrical, and had obtuse to slightly rounded ends. The size of conidia was (11.6-)13–15(−16.2) × (4.1-)4.6–5.6(−6) μm. Appressoria were brown, ovoid to ellipsoid or slightly irregularly to irregularly shaped, and the average size was 6.8–9 × 5.1–6.5 μm. Pathogenicity of the isolates to fruits and leaves were compared, and genes from all six isolates were sequenced. The isolates were identified as C. siamense based on four-gene phylogenetic analyses (ribosomal DNA-ITS, GAPDH, ACT and CHS-1) and morphological as well as cultural characters. This is the first report of C. siamense as a causal agent of anthracnose of walnut in China.