The hairy roots of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey. are capable of producing active compounds such as iridoid glycoside, and phenylethanoid glycosides, which have potential applications in the pharmaceutical industry. Silver nanoparticles (AgNPs) have been used as novel elicitors in the induced cultivation of hairy roots, but there is a lack of research regarding their effects on R. glutinosa hairy roots. In the present study, silver nanoparticles (Pp-AgNPs) synthesized by the endophytic fungus Penicillium polandii PG21 were adopted to elicit hairy roots of R. glutinosa, to investigate their influences on the biomass, color, secondary metabolites, antioxidant activity, sucrose metabolism, and phytohormone-related gene expression. The results revealed that the dry weight and fresh weight of R. glutinosa hairy roots were both higher in the treated group than in the control group after addition of 2 mg/L Pp-AgNPs for 20 d. The content of verbascoside, total phenylethanol glycosides and total cycloartenoid in the treatment group reached the highest level at 20 d, which were 1.75, 1.51, 1.44 times more than those in the control group, respectively. Pp-AgNPs significantly stimulated the enzyme activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). The growth-promoting effect of Pp-AgNPs may be accomplished by increasing sucrose metabolism, and regulating the synthesis and signal transduction of gibberellin (GA) and indoleacetic acid (IAA). Moreover, expressed sequence tags-simple sequence repeat (EST-SSR)-based genetic diversity analyses indicated that there was little possibility of genetic variation among samples under different treatment conditions. In conclusion, the appropriate concentration of Pp-AgNPs can be used as an effective elicitor to improve the biomass and secondary metabolites content in R. glutinosa hairy roots.