Lady Lilith (1866-1868, altered 1872-1873) belongs to Rossetti's famous series of half-length female portraits, which started with the painting Bocca Baciata (1859). The subject of the painting originates from Jewish mythology: Lilith was the first wife of Adam, and unlike Eve, she was created at the same time and from the same clay as Adam. In one version of the legend, Lilith betrayed Adam, after refusing to be his subordinate, and fled the Garden of Eden to join the fallen angel Samael. She is a demonic figure and seductress, known in folklore stories for killing babies and bewitching men.

In a letter from 1870 to his friend Dr. Hake, Rossetti wrote about the painting: "The picture… represents a Modern Lilith combing out her abundant golden hair and gazing on herself in the glass with complete self-absorption by whose fascination such natures draw others within their circle." Rossetti wrote the sonnet Body's Beauty that accompanies the painting. In the sonnet, he describes Lilith the seductress, who entraps young men with the strains of her golden hair. Rossetti represents Lilith's dangerous nature through her luscious hair, as well as the paraphernalia surrounding her. The roses, a flower associated with Aphrodite, symbolize sensuality, while the poppies signify sleep and forgetfulness. On the dresser under the mirror is a spray of foxglove, which can be used as a medicine, but also as a deadly poison. The painting forms a pendant with Sibylla Palmifera (ca. 1865-1870) that was accompanied by Rossetti's sonnet Soul's Beauty. The two figures embody contrasting ideas: Lilith represents bodily sin and vice, while Sibylla signifies the ideal of female virtue.

Scholars have suggested that Lady Lilith was inspired by Titian's painting Woman with a Mirror (1515). Rossetti saw the painting during his trip to Paris in 1864, where he spent his time exploring the Louvre's collection. The two paintings share several pictorial elements: Rossetti emulated Titian's treatment of the female's flesh, as well as the central parting and the wavy curls of the cascading hair. Additionally, Rossetti referred to Lady Lilith as a 'toilette painting,' reinforcing the assumption that Titian's work influenced him. The painting is also linked to the portrait Jo, the Beautiful Irish Girl (1865) by French Realist Gustave Courbet. It is possible that Courbet was inspired by Rossetti, who visited his studio during his stay in Paris in 1864.

Rossetti worked on the painting for several years and created at least four related drawings of the composition, such as Study for Lady Lilith (1886), as well as watercolor replicas like Lady Lilith (1867). The model in the preparatory drawings, watercolors, and the original oil painting is Fanny Cornforth, Rossetti's housekeeper, and mistress. The painting was commissioned by Rossetti's patron Frederick Leyland, who received the picture in the spring of 1869. However, in 1872 Leyland asked Rossetti for a significant alteration: he wanted to substitute Cornforth's face with that of Alexa Wilding, who Leyland felt had more 'refined' features. It remains unclear whether Rossetti agreed with Leyland's assessment and whether he was ultimately pleased with the outcome.