Soil acidification significantly impacts peanut (Arachis hypogaea L.) yield in China. The objective of this study was to investigate the effects of the physiological mechanisms of soil acidification on the growth and development of peanut seedlings. A pot experiment was conducted to investigate the effects of soils with varying pH levels (pH6, pH4, and pH3) on root morphology, leaf photosynthesis, chloroplast ultrastructure, nutrient and dry matter accumulation of peanut seedlings (Huayu 22 and Huayu 20, abbreviated as HY22 and HY20). In comparison to the control soil with a pH of 6, soil with a pH of 3 inhibited the growth of fine roots in peanut but promoted the growth of thick roots. And the soil with a pH of 3 also reduced the leaf photosynthetic rate (Pn) by decreasing stomatal conductance (Gs), SPAD value and chlorophyll fluorescence indices, including maximum quantum yield (Fv/Fm), electron transport efficiency (ETR) and PSII actual photochemical quantum yield (Y). Morphology and ultrastructure of chloroplast were altered by in response to pH3 stress, such as a decrease in number of chloroplasts per cell and number of grana per chloroplast, an increase in osmiophilic particles per chloroplast, dissolution of cell membranes and chloroplast outer membranes. The effects of soil with a pH of 4 on the morphology and physiological indicators of peanut seedlings were less pronounced than those observed in soil with a pH of 3. Soil with a pH of 3 led to poor root morphology, destruction of leaf photosynthetic system and abnormal morphology and reduced function of chloroplasts, ultimately resulting in decreased dry matter accumulation. In soil with a pH of 4, peanut seedlings were able to maintain nearly normal growth. These findings enhance our understanding of the morphological and physiological mechanisms underlying peanut responses to low pH.