This study aims to design and synthesize Endostatin (ENT)-loaded nanoparticles using Folic acid (FA) as a driver for targeted anti-proliferative chemotherapy in Esophageal Squamous Cell Carcinoma (ESCC). An ionic gelation method was employed to formulate FA-decorated, ENT-loaded nanoparticles, which were tested in vitro on KYSE-30 cells using unbiased stereological approaches. FA-ENT nanoparticles were internalized into ESCC cells with preferential binging to the nucleus and mitochondria for necrotic and apoptotic effects. Nanoparticles showed increased proliferation inhibition of 64.71% and reduced KYSE-30 cell migration of up to 74.12% when compared to the control. Positively charged spherical nanoparticles, with selective pH responsive ENT release, were further tested in vivo employing a tumor xenograft model. Tumor mass increased up to 5505.54 mm³ in the control group while a substantial reduction occurred in the treatment group (native ENT, ENT-nano and FA-ENT-nano) down to 128.23 mm³ (97.67%). Tumor volume was reduced from 1000.2 mm³ to 567.64 mm³ (43.25%) in the native ENT group, from 324.43 mm³ to 190.25 mm³ (41.36%) in ENT-nano group (non-targeted system), and from 1374.21 mm³ to 998.67 mm³ (27.33%) in FA-ENT-nano group (targeted system) following treatment. There were no significant differences in the body weight of mice treated with the nano-formulations as opposed to the control mice. FA-decorated nanoparticles for active transport of ENT into tumor cells with an enhanced in vitro and in vivo anti-proliferative efficacy in ESCC therapy were synthesized.