Acquired chemoresistance refers to tumor cells gradually losing their sensitivity to anticancer drugs during the course of treatment, resulting in tumor progression or recurrence. This phenomenon, which has deleterious outcomes for the patient, has long been observed in patients with glioblastoma receiving temozolomide (TMZ)-based radiochemotherapy. Currently, the mechanisms for acquired TMZ chemoresistance are not fully understood. In the present study, a TMZ-resistant cell line U251R with a 4-fold 50% inhibition concentration compared with its TMZ-sensitive parent cell line was isolated by incremental long-time TMZ treatment in the human glioblastoma cell line U251. Fluorescence-activated cell sorting analysis indicated G2/M arrest and a lower proportion of cells in the S phase, accompanied by a decreased apoptosis rate in the U251R cell line compared with the parental U251 cell line. In addition, a sphere-formation assay indicated an increased self-renewal capacity in U251R cells. Furthermore, a high-throughput protein microarray unveiled more than 200 differentially expressed proteins as potential molecular targets accounting for acquired TMZ resistance. Subsequent bioinformatics analysis illustrated the molecular and signaling networks and revealed the central role of SRC. Immunoblotting and reverse-transcription quantitative polymerase chain reaction analysis further confirmed the expressional upregulation of SRC family kinases. Moreover, SRC knockdown led to partial reversal of TMZ resistance in the U251R cell line and sensitization in the U373 cell line. These data helped to develop a comprehensive understanding of survival strategies, particularly with respect to pro-stemness regulation, which could be potential targets for overcoming TMZ resistance.