Localized drug delivery strategies for cancer therapy have been introduced for decades as a means of increasing drug concentration at tumor target site and minimizing systemic toxicities. In this paper, a combination of microspheres (MSs) and sucrose acetate isobutyrate (SAIB) in situ-forming implants (ISFIs) was evaluated for improving antitumor efficacy via intratumoral injection. Monodispersed cucurbitacin (Cuc)-loaded Poly (lactic-co-glycolic acid) (PLGA) MSs with mean diameter of about 5 μm were fabricated by Shirasu porous Glass (SPG) membrane emulsification technique, and their properties were investigated. The in vitro drug release pattern, antimelanoma efficiency, and drug distribution in tumor of three different intratumoral injection systems, that is, MSs, SAIB ISFIs, and combination of MSs and SAIB ISFIs (SAIB-MSs), was investigated. The Cuc-loaded MSs prepared by PLGA (LA/GA = 50:50, inherent viscosity = 0.87 dL/g), has an appropriate release pattern with lower initial burst and delayed drug release. SAIB-MSs have a much slower drug release rate than that of MSs or SAIB ISFIs. SAIB-MSs showed the best antitumor efficacy in melanoma-bearing mice model, and the results of drug distribution in tumor revealed that the incorporation MSs in SAIB solution obviously extended the residence of drug in tumor. The low Cuc concentration in tumor periphery region after intratumoral administration of SAIB-MSs demonstrated poor drug penetration of this system. For further improving the antitumor efficacy of intratumoral chemotherapy, elegant designing to carriers with both extended residency and wide drug distribution in tumor is needed.