Building on our prior research, a novel series of trimethoxyphenoxymethyl- and trimethoxybenzyl-substituted triazolothiadiazine compounds has been designed and achieved successfully via a direct ring-closing strategy. Initial biological evaluation illustrated that the most active derivative B₅ exhibited significant cell growth inhibitory activity toward HeLa, HT-29, and A549 giving the IC₅₀ values of 0.046, 0.57, and 0.96 μM, respectively, which are greater or similar with CA-4. The mechanism study revealed that B₅ caused the G₂/M phase arrest, induced cell apoptosis in HeLa cells in a concentration-dependent manner, and also showed potent tubulin polymerization inhibitory effect. Meanwhile, B₅ exerted significant antivascular activity in the wound-healing and tube formation assays. Most importantly, B₅ remarkably inhibited tumor growth without obvious signs of toxicity in A549-xenograft mice model. These observations indicate that 6-p-tolyl-3-(3,4,5-trimethoxybenzyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine might be considered as the potential lead compound to develop highly efficient anticancer agents with potent selectivity over normal human cells.